ES2387280A1 - pTOP: NEW VECTOR OF EXPRESSION AND PURIFICATION OF PROTEINS. - Google Patents

Abstract

pTOP: New protein expression and purification vector. The present invention refers to (1) a new gene expression vector that allows protein labelling, expression and purification, (2) to the associated protein expression and purification method to this vector and (3) to a kit comprising said vector.

Description

pTOP: NEW PROTEIN EXPRESSION AND PURIFICATION VECTOR

The present invention belongs to the field of Molecular Biology, Genetics and Biotechnology. The present invention relates to (1) a new gene expression vector that allows for the labeling, expression and purification of proteins, (2) to the method of protein expression and purification associated with this vector and (3) to a kit that It comprises said vector.

STATE OF THE PREVIOUS TECHNIQUE

There are numerous examples of expression vectors on the market, among which are GE Healthcare Life Sciences pGEX vectors or Invitrogen pBAD vectors. In these two series of vectors the cloning of the gene of interest can be carried out by restriction. In addition, there are commercial pBAD vectors prepared to clone by using topoisomerase, or using donor vectors for recombination with input pBAD vectors by Gateway recombination technology. Both systems allow controlling the expression of the gene of interest thanks to two operons, the lac (lactose) operon that is induced in the presence of lactose or also of IPTG (isopropyl- ~ -D-1-thiogalactopyranoside, a lactose analogue but more advantageous because its levels are maintained because it does not degrade during cell culture) in cultures of cells transformed with pGEX, and the ara operon (of arabinose) that is induced by the addition of arabinose in cultures of cells transformed with pBAD. In addition, both systems allow labeling and obtaining proteins with different markers, such as fusion with GST (Glutathione S-transferase), thioredoxin, a chain of 6 histidines, the V5 epitope (the long form consists of 14 amino acids and the 9 amino acid cut and are derived from the Pk epitope present in the P and V proteins of the apex 5 paramyxovirus (SV5)), or the myc epitope (10 amino acids and is part of the transcription factor c sequence -myc human), which facilitate both the detection and purification of the expressed protein. These vectors also allow the processing of the resulting protein with specific proteases to thereby release the protein of interest, such as the Pre-Scission protease (GST fusion protein with the human rhinovirus HRV 3C protease (HRV) type 14), the protease Thrombin (purified from bovine plasma) or Factor Xa protease (purified from bovine plasma), in the case of the pGEX vector, or Enterokinase protease (purified from bovine intestine or using the recombinant catalytic subunit EKMax ™ Enterokinase marketed by Invitrogen) in the pBAD case.

These vectors allow obtaining the protein of interest fused with a marker or free. The proteins fused to the markers carry, in addition to the corresponding sequence encoded by the DNA of the gene of interest and the markers, other amino acid residues added not belonging thereto, but are those encoded by the cut-off targets of the proteases and by the restriction targets or recombination sites used in cloning. Although in smaller numbers, after release of the proteins of interest by cutting with specific proteases, some of these residues remain attached to their sequence. Since PreScission and Thrombin only leave 2 residues after cutting, and Factor Xa and Enterokinase cut exactly at the C-terminal end of their target, most of the remaining residues after cutting are mainly due to those encoded and introduced by the sequences of the restriction targets and / or the specific sites necessary for cloning.

Stratagene's p-CAL-n-FLAG vector is another example of an expression vector that allows obtaining a fusion protein with different elements, consisting of the CBP peptide ("calmodulin-binding peptide" or binding peptide) to calmodulin), the thrombin protease target amino acid sequence, the FLAG marker (unnatural 8 amino acid epitope designed specifically as a protein marker) and the Enterokinase protease target amino acid sequence, all these elements being located at the amino terminal end of The protein of interest. The gene of interest is cloned by an LlC ("Iigation independent cloning ') method, which, unlike other methods used in cloning in pGEX and pBAD, prevents the addition of extra residues from the cloning stage to the protein of The elements presented by the fusion protein resulting from expression in the p-CAL-n-FLAG vector have different uses: CBP allows affinity purification of the fusion protein in a calmodulin resin. The FLAG marker is an epitope recognized by specific antibodies, which also allows the entrapment of the fusion protein, either for purification or for example for the study of protein-protein or protein-DNA interactions by immunoprecipitation or immunoprecipitation of chromatin. release the protein thus obtained from the CBP marker by cutting with the thrombin protease, since in this type of immunoprecipitation studies the presence CBP can be counterproductive, since it can be nonspecifically bound to many calmodulin-binding cellular proteins, so it is convenient to remove it to obtain the protein of interest fused only to the FLAG marker and the Enterokinase target.

DESCRIPTION OF THE INVENTION

In a first aspect, the present invention provides a new vector, called pTOP (Figures 1 and 2), for gene expression and protein purification based on a nucleotide sequence containing different elements. This vector allows the labeling of the protein of interest with different labels or markers, which facilitate its manipulation and purification, and also allows the obtaining of the protein of interest in different forms: fused with several markers, with a single marker or pure protein , without any extra amino acid (Figure 3).

More specifically, the expression vector of the invention allows obtaining a fusion protein with three markers and two target protease sequences arranged in such a way that the protein of interest can be purified fused with all these elements, with a single marker or simply the sequence of the protein of interest (Figure 3), in a single stage or in several stages sequentially.

The markers of the fusion protein that is obtained thanks to the expression vector of the invention have several advantages, such as that they allow the purification of the affinity fusion protein in different types of systems, which increases the versatility of the purification method and makes it therefore more efficient. The fusion protein carries GST and, therefore, can be purified by glutathione affinity or by immunoprecipitation, since there are specific antibodies against GST. In addition, the fusion protein carries two polyhistidine epitopes, which allows its purification by affinity for metals such as nickel or cobalt, or by immunoprecipitation, since there are specific antibodies against polyhistidine epitopes. The fact that the protein carries two polyhistidine epitopes instead of one, as in other cases of the prior art, allows the purification to be more efficient, since the protein binds with more affinity to the metal and this allows it they can perform more washes and greater astringency to selectively remove other unwanted cellular proteins that also exhibit affinity for the metal.

Another advantage of the fusion protein obtained by the expression pTOP vector of the invention is that it can be purified in different ways and in successive steps, such as a first purification of the fusion protein containing all the elements in a nickel column. and elution with imidazole, a second purification on a glutathione column, which can be followed by processing the protein with the PreScission protease to release it from GST and the first polyhistidine and then a third affinity purification by antibodies against The second polyhistidine.

The vector of the first aspect of the invention also has the advantage that it comprises an element that allows the control of the expression of the protein of interest by the addition of a substance, that is, it has an inducible promoter.

In a second aspect, the present invention provides a new method for gene expression and protein purification using the vector of the first aspect of the invention. Thanks to the characteristics of the vector of the first aspect of the invention, the purification of the protein expressed by said vector is very flexible, since it can be carried out, as desired, in a single stage or in several, depending on the needs of purity or of the difficulties of obtaining each protein, and if it is desired to purify the labeled protein with one or two markers to be recognized by specific antibodies or by other molecules.

In a third aspect, the present invention provides a kit comprising the vector of the first aspect of the invention, as well as instructions for carrying out the cloning of the gene, inducing the expression of the corresponding protein and its purification by the method. of the second aspect of the invention.

Therefore, a first aspect of the invention relates to a nucleotide sequence comprising the following elements:

to.

an initiation codon,

b.

a nucleotide sequence encoding an M marker,

C.

a nucleotide sequence encoding an N marker,

d.

a nucleotide sequence encoding a protease recognition amino acid sequence P,

and.

a nucleotide sequence encoding a 0 marker,

F.

a nucleotide sequence encoding a protease recognition amino acid sequence Q,

g.

a termination codon.

In a preferred embodiment of the first aspect of the invention, the nucleotide sequence comprises a sequence consisting essentially of the following elements:

to.

an initiation codon,

b.

a nucleotide sequence encoding an M marker,

C.

a nucleotide sequence encoding an N marker,

d.

a nucleotide sequence encoding a protease recognition amino acid sequence P,

and.

a nucleotide sequence encoding a 0 marker,

F.

a nucleotide sequence encoding the amino acid sequence Q of recognition of a protease,

g.

a termination codon.

An embodiment of the first aspect of the invention may be a nucleotide sequence consisting of elements (a) - (g) and that has some other characteristic that is not relevant to the function of the first aspect of the invention. For example, a non-relevant feature could be a multi-cloning region (MCS), which consists of the succession of specific targets of restriction enzymes. This element would not be relevant since it would not imply any advantage for the expression of the protein of interest, and it is also not essential for the cloning of said protein of interest in the nucleotide sequence of the invention, since they exist in the state of the art. other ways to clone a DNA fragment.

In the examples herein the inventors use a new method of cloning by CiPCR (reverse cloning PCR), which comprises a classical PCR of amplification of the insert and then the cloning of this by means of a CiPCR.

In a more preferred embodiment of the first aspect of the invention, the nucleotide sequence comprises the elements in the following order: (a) an initiation codon; (b) a nucleotide sequence encoding an M marker; (e) a nucleotide sequence encoding an N marker;

(d) a nucleotide sequence encoding a protease recognition amino acid sequence P; (e) a nucleotide sequence encoding an O marker; (f) a nucleotide sequence encoding an amino acid sequence Q for recognition of a protease; (g) a termination codon.

In a preferred embodiment of the first aspect of the invention, the M and O markers are the same. In a preferred embodiment of the first aspect of the invention, the M, NYO markers are selected from the list comprising: polyhistidine, GST, avidin, streptavidin, HA, VSV-G, HSVtk, FLAG, maltose binding protein, V5, myc and a fluorescent protein. Preferably, the M, N and O markers are selected from polyhistidine and GST. More preferably, the M and O markers are a polyhistidine and the N marker is the GST. Even more preferably, the M and O markers are a polyhistidine between 6 and 14, preferably 10 histidines.

The HA or hemagglutinin marker peptide is an epitope found in the human influenza virus, VSV-G belongs to the vesicular stomatitis virus glycoprotein, the HSVtk marker peptide belongs to the herpes simplex virus type 1 Thymidine Kinase, and the rest have been described above.

The term "marker", as used herein, refers to a marker peptide or marker protein, which allows the identification of the protein of interest when produced together with said protein as a fusion protein. The marker peptide or the marker protein serves to identify and / or locate and / or trap the protein of interest because they are binding sites to certain molecules or atoms, or because they are easily detectable by immunochemical techniques, both polyhistidine, GST , avidin or streptavidin, such as HA, VSV-G, HSVtk, FLAG, V5 or myc, or because they are also easily observable by other methods, as with fluorescent proteins.

In a preferred embodiment of the first aspect of the invention, the P and Q protease recognition amino acid sequences are selected from the list comprising the protease targets: Pre-Scission, Enterokinase, Thrombin, Tev, HRV 3C, ULP1 and factor For. Preferably, the amino acid sequence P is the recognition target of the Pre-Scission protease, and the amino acid sequence Q is the recognition target of the protease Enterokinase.

The Tev protease is a protease of the Tobacco Etch Virus. The HRV 3C protease is a 3C protease of human rhinovirus type 14. The ULP1 protease ("Ubl-specific protease 1 ') is a protease of the Ubls family (" ubiquitin-like proteins'), of the yeast Saccharomyces cerevisiae ( S. Cerevisiae), which is involved in the rupture of SUMO ("small ubiquitin-like modifier") to release it from its substrates.

The term "protease", as used herein, refers to an enzyme capable of breaking the peptide bond between two determined amino acids within a specific amino acid sequence, which is the target (recognition) sequence of said protease. .

In a preferred embodiment of the first aspect of the invention, the nucleotide sequence further comprises an inducible promoter. Preferably, the inducible promoter is selected from the list comprising: arabinose inducible promoter, lactose or IPTG inducible promoter, ecdysone inducible promoter, tetracycline inducible promoter, galactose inducible promoter, heavy metal inducible promoter, mifepristone inducible promoter , tryptophan inducible promoter, temperature inducible promoter, phosphate inducible promoter and methanol inducible promoter. More preferably, the promoter is arabinose inducible.

A promoter is a nucleotide sequence that controls the transcription of the gene of interest. The term "inducible," as used herein, refers to the possibility of controlling the expression of a nucleotide sequence over time, and refers to the presence of a control element. Said control element allows the expression to be activated or deactivated by, for example, the addition of a substance to the culture medium. An arabinose inducible promoter is one that has the necessary elements for transcription to occur only in the presence of this substance. An embodiment of the first aspect of the invention may be an arabinose inducible promoter comprising the araC gene and the pBAD promoter of the Escherichia coli arabinose operon (E. coli).

In a preferred embodiment of the first aspect of the invention, the nucleotide sequence further comprises at least one origin of replication. The origin of replication can be from prokaryotes or eukaryotes. Preferably, it comprises the origin of replication of plasmid pUCo

The term "origin of replication", as used herein, refers to a nucleotide sequence where a replication fork is formed and where DNA replication begins.

In another preferred embodiment of the first aspect of the invention, the nucleotide sequence further comprises a selection gene. Preferably, the selection gene is chosen from the list comprising: an antibiotic resistance gene, a gene that codes for a fluorescent protein, a gene that codes for a luminescent protein, a gene that codes for betagalactosidase and a gene that converts auxotrophic strains into prototrophs. More preferably, the selection gene is an antibiotic resistance gene. Preferably, the antibiotic is ampicillin, kanamycin, tetracycline, chloramphenicol, geneticin, hygromycin B, puromycin, blasticidine, methotrexate or zeocin. More preferably, it is an ampicillin resistance gene.

The term "selection gene", as used in the present description, refers to a gene that encodes a protein that confers a distinctive characteristic to the organism in which it is expressed, as it might survive the presence of an antibiotic, which produces a colored substance in the presence of a specific reagent or that emits light. An antibiotic resistance gene codes for a protein that confers on the cell that expresses it, which would normally be sensitive to the antibiotic, the ability to overcome the antibiotic's effect. Said protein is usually an enzyme that inactivates or expels the antibiotic in question.

In a preferred embodiment of the first aspect of the invention, the nucleotide sequence is SEO ID NO: 1 or a bioequivalent variant. The term "bioequivalent variant", as used in the present description, refers to a sequence that has the same elements as SEO ID NO: 1 and which, although it may have a slightly different sequence, retains the functional characteristics of SEO ID NO: 1, that is, the differences with the SEO sequence ID NO: 1 are not relevant.

Preferably, the nucleotide sequence of the first aspect of the invention is a gene construct or is part of a gene construct. More preferably, it is a gene construct. A gene construct is a nucleic acid molecule in which different elements have been arranged in a specific and desired way. These elements can be, among others, replication sequences, control sequences, coding sequences, multicloning sequences or recombination sequences.

Preferably, the nucleotide sequence of the first aspect of the invention is a vector or part of a vector. More preferably, it is a vector. A vector is a gene or DNA or RNA sequence construct that contains a series of functional elements, including an origin of replication, control sequences, such as, for example, translation control elements (such as initiation and translation codes). stop) and transcription (for example, promoter-operator regions, binding sites of transcription factors or regulatory elements). The vector may include bacterial plasmids, viral vectors and other vectors well known and documented in the state of the art. A variety of techniques that can be used to introduce such vectors into prokaryotic or eukaryotic cells (host cells) for expression are also known. Appropriate transformation or transfection techniques are well described in the state of the art. A replicon is therefore any genetic element that behaves as an autonomous unit of gene replication within a cell; that is, able to replicate under its own control. A vector is therefore a replicon that, by cloning another polynucleotide segment, allows replication and / or expression of the bound segment. The term "control sequence" refers to nucleotide sequences that are necessary to effect the expression of the coding sequences to which they are linked. The nature of such control sequences differs depending on the host organism; in prokaryotes, said control sequences generally include a promoter, a ribosomal binding site, and termination signals; in eukaryotes, generally, said control sequences include promoters, termination signals, enhancers and, sometimes, silencers. The term "control sequences" is intended to include, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous.

A second aspect of the invention relates to a method of gene expression and protein purification, characterized in that it comprises a first stage (h):

h. Clone a gene of interest in a nucleotide sequence, gene construct or vector of the first aspect of the invention.

The term "cloning", as used herein, refers to the process that allows the generation of copies of a DNA fragment, such as the insertion of a nucleotide sequence of interest into a vector that can be replicated. within a cell The cloning of step (h) is carried out so that the sequence of the gene of interest is functionally linked to elements (a) - (g) of the first aspect of the invention. The gene of interest is inserted between the elements (f) and (g), originating a coding region comprising (a), (b), (c), (d), (e), (f) and the gene of interest. Therefore, the insertion site ("i") of the sequence of interest is exactly between the nucleotide sequence encoding the amino acid sequence Q of recognition of a protease and the termination codon, and is represented in the figures of this description as ** --_ ## or ** i ##.

The term "functionally linked" refers to the fact that the reading pattern, in the process of translating the genetic code from the nucleic acid to the protein, is adequate for the fusion protein that presents the elements (a) to be produced - (f) at its amino terminal end, and then the amino acid sequence of the protein of interest.

The gene of interest (represented in the figures as "gene") is the nucleotide sequence of interest inserted in ** i ## (Figure 2), and which codes for the protein of interest. The term "protein of interest", as used herein, refers to any protein whose coding sequence is known, available or will be available, so that it can be cloned into an appropriate vector for expression in the appropriate cells or in an in vitro expression system.

Cloning of the sequence of interest can be carried out by the cloning methods described in the state of the art. However, not all known cloning methods allow to obtain the pure protein without any extra amino acid. Therefore, the inventors recommend the use of the CiPCR cloning method, which in addition to allowing the different variants of the fusion protein to be obtained (Figure 3), also allows obtaining the pure protein without extra amino acids, and also has other advantages such as: 1) simplifying the cloning process, 2) not requiring high knowledge of genetic engineering, 3) avoiding the laborious design and implementation of a cloning strategy based on the presence of specific targets of restriction or recombination enzymes.

This new method of cloning by CiPCR is based on the design of two direct and reverse primer oligonucleotides, whose respective 3 'ends hybridize with the sequence of the gene of interest to be inserted, for priming and amplification. After amplification of the gene of interest with these primers, both the 5 'and 3' ends of each of the amplicons obtained hybridize with each of the pTOP vector chains in a second reaction, the CiPCR.

For the design of these oligonucleotide primers, the direct oligonucleotide begins with the sequence 5'-cacgatgacgatgacaag-3 '(SEO ID NO: 22), and then the sequence of the direct primer designed for the amplification of the gene of interest to be cloned is added without the initiation codon, obtaining the complete direct oligonucleotide: 5'cacgatgacgatgacaag (SEO ID NO: 22) + sequence-primer-direct-gene-interest 3 '. The reverse oligonucleotide begins at the 5 'end with the sequence 5'-atgacaactccgtcttcc-3' (SEO ID NO: 23) and then the reverse primer sequence designed for amplification of the gene of interest is added without including the stop codon, since this is included in the part corresponding to the sequence of the pTOP vector, obtaining the complete reverse oligonucleotide: 5'atgacaactccgtcttcctta (SEO ID NO: 23) + sequence-primer-reverse-geninterés 3 '.

The two hybridization sequences with the direct and reverse primer vector (SEO ID NO: 22 and SEO ID NO: 23) correspond to the sequences of the pTOP vector flanking the insertion site of the gene to be cloned ** --_ ## . In SEO ID NO: 1, which corresponds to the empty pTOP vector, its 3 'ends coincide with positions 1,093 and 1,094 respectively.

In the oligonucleotide primers, it may be of interest to add optional M mutations in two regions: -between the sequence that hybridizes with the vector and the priming sequence for the amplification of the gene of interest to be inserted into pTOP-within the own primer sequences designed for amplification of the gene of interest to be inserted or cloned.

With the first ones, a new label can be inserted, both at the amino terminal end after the EK target, and at the carboxyl terminal before the stop codon. With the latter, M mutations can be introduced at sites near both the amino terminal and the carboxyl terminal of the protein of interest.

In a first conventional PCR, these oligonucleotide primers are used to amplify the sequence of interest from the template available or to amplify and mutate it. In a second PCR, called CiPCR, the circular pTOP vector is used as a template and the double-stranded amplicons product of the first conventional PCR are used as primers, where each amplicon chain of the first conventional PCR hybridizes at its 5 'and 3' ends simultaneously with the corresponding pTOP vector chain and acts as a primer (at its 3 'end) and an elongation terminator (at its 5' end) in the CiPCR.

Alternatively to the double-stranded amplicons of the first conventional PCR, the double-stranded amplicons obtained after the reamplification of said amplicons can be used with direct and reverse priming oligonucleotides, which allow the size of the hybridization sequence of the 5 'and 3 ends to be extended. 'of the amplicon with the sequence of the pTOP vector at the place of insertion of the sequence of interest to be cloned. Such primers may have a variable length from a minimum of 30 to a maximum of 135 nucleotides, preferably between 30 and 50 nucleotides.

The sequence of the direct reamplification primer matches that of a fragment of the pTOP vector from the nucleotide at position 1.093 of SEO ID NO: 1, which would be the nucleotide of the 3 'end of the direct primer. For example, a direct reamplification primer for cloning by CiPCR in 37 nucleotide pTOP would have the following sequence (SEO ID NO: 20): 5'tcaccatcaccatcaccatcacgatgacgatgacaag3 ', coinciding with the sequence of the pTOP vector (SEO ID NO: 1) from the nucleotide at position 1.057, which would be the nucleotide of the 5 'end of the direct primer, to the nucleotide at position 1.093, which would be the nucleotide of the 3' end of the direct primer.

The reverse reamplification primer sequence is complementary to that of pTOP and its 3 'end would be the nucleotide complementary to the nucleotide at position 1,094 of SEO ID NO: 1. An example of the reverse nucleotide primer could be 40 (SEO ID NO: 21): 5'gcccaagcttcgaattccaatgacaactccgtcttcctta3 ', whose sequence is complementary to that of the pTOP chain indicated in SEO ID NO: 1, so that its 3' end would be the nucleotide complementary to the nucleotide at position 1.094 and its 5 'end would be the nucleotide complementary to that of position 1 .133 of SEO lONa: 1.

To avoid a second conventional insert reactivation PCR with the primers described above, the pair of primers designed for the first insert amplification PCR may already carry hybridization sequences with the vector of greater length than those described above (SEO ID NO : 22 and SEO ID NO: 23) at its 5 'ends.

The use of amplicons with a high number of residues in the sequences of their ends for hybridization with the vector, obtained either by reamplification of the amplicons of the conventional PCR of amplification of the insert or using directly in this oligonucleotide primers with a greater number of residues , can result in a significant increase in cloning efficiency. A study of the melting temperatures, the formation of dimers, and other parameters of the oligonucleotides that include the hybridization sequences with the insert and with the vector and the M mutations where appropriate, will help in the choice of the strategy to be followed.

The CiPCR, due to the characteristics derived from the use of circular molds and primer-terminator sequences (whose properties derive from the special design of the direct and reverse oligonucleotides), allows 1) to clone in a directed manner, 2) to insert inserts between the ends of the insert and of the vector and / or mutations (insertions, deletions or substitutions) at the ends of the vector and / or of the insert simultaneously to the cloning, 3) obtain complementary linear amplicons but with long complementary cohesive ends also with each other, 4) these amplicons , unlike what happens in a conventional PCR, they are characterized because they cannot be used as templates in the CiPCR, and because they are stabilized by in situ hybridization giving rise to a vector type gene construct that has a single cut in each of their two chains and they carry the insert

or the insert and the desired insertions and / or mutations incorporated in the desired place (s).

The starting template vector molecules of the CiPCR are destroyed by a methylation-dependent endonuclease, and the resulting circularly stabilized vector-type gene constructs are introduced into a prokaryotic cell, preferably E. coli, where the cell ligases covalently join the cut. present in each of the chains, and allow the vector to replicate and inherit during cell division for amplification and subsequent extraction, purification and analysis of the corresponding plasmid.

In a preferred embodiment of the second aspect of the invention, after step (h) the following step (i) is carried out:

i. Produce the protein encoded by the sequence cloned in step (h).

In a preferred embodiment of the second aspect of the invention, step (i)

It comprises at least two sub-stages:

i '. Enter the nucleotide sequence, gene construct or vector

obtained in step (h) in a prokaryotic cell or in a cell

eukaryotic,

i ". Cultivate the cell of stage (i ') in the presence of substances

necessary for the protein encoded by the gene to be expressed

cloned interest in stage (h).

Preferably, in step (i ') the nucleotide sequence, gene construct or vector is introduced into an E. coli cell. More preferably it is

E. coli BL21. Preferably, in step (i ') the nucleotide sequence, gene construct or vector is introduced into a yeast cell, an insect cell or a mammalian cell. More preferably, the yeast cell is S. cerevisiae or Pichia pastoris (P. pastoris). Examples of insect cells are the SF9, SF21, High-5 or Mimic-SF9 lines. Examples of mammalian cells are, but are not limited to, lines of tumor origin such as the Cos, JEG3, NCI-H460, Jurkat, PC12 lines.

The E. coli bacterium belongs to Prokaryota Super Kingdom, Bacteria Kingdom, Phylum Proteobacteria, Gammaproteobacteria Class, Enterobacterial Order, Enterobacteriaceae Family and Escherichia Genus.

The brewer's yeast S. cerevisiae is a unicellular fungus that belongs to Superuk Eukaryota, (Metazoa / Fungi group), Fungi Kingdom, Subreino Dikarya, Phylum Ascomycota, Subphylum Saccharomycotina, Class Saccharomycetes, Order Saccharomycetales, Family Saccharomycetaceae and Genus Saccharomycetaceae and Genus Saccharomycetaceae

Yeast P. Pastoris is a unicellular fungus that belongs to Superuk Eukaryota, (Metazoa / Fungi group), Kingdom Fungi, Subreino Dikarya, Phylum Ascomycota, Subphylum Saccharomycotina, Class Saccharomycetes, Order Saccharomycetales, Family Saccharomycetaceae and Genus: Pichia.

In a preferred embodiment of the second aspect of the invention, the cultivation of step (i ") is done in the presence of arabinose.

In a preferred embodiment of the second aspect of the invention, after step (i) the following step (j) is carried out:

j. purify the protein produced in step (i).

Preferably, step U) comprises at least three sub-stages: j '. List the cultured cells in step (i "). J". Bind the lysate protein obtained in step (j ') to a ligand immobilized on a solid support. j "'. Release the protein of stage U") from its binding to the ligand of stage (j ").

Preferably, the immobilized ligand on a solid support of step (j ") is glutathione. Preferably, the immobilized ligand on a solid support of step (j") is protein A, protein G or a metal ion, more preferably it is a metal ion Preferably, the metal ion is nickel (11) (Ni) and / or cobalt (11). The binding of the protein to the ligand can also be through an anti-polyhistidine antibody and / or an anti-GST antibody that binds to protein A or G protein ligands immobilized on a solid support, preferably agarose or sepharose. The stages j "and j '" can be repeated sequentially, so that different immobilized ligands can be used on a solid support for sequential purification of the protein in different stages.

The term "Iisar", as used herein, refers to the action of breaking down cell walls and / or membranes to release cellular content.

The solid support on which the ligand is immobilized can be, for example, but not limited to, an acrylic resin, agarose or sepharose balls, iron shavings (Fe), which can be presented in suspension or in a column, a cartridge, a plate, a magnetizable wire or a magnet, among others.

In a preferred embodiment of the second aspect of the invention, the protein is released from its binding to the immobilized ligand by at least one of the following ways: a change in pH, the addition of free ligand, the addition of a molecule that moves to the protein by competition for ligand binding, treatment with a reducing agent, processing with a protease. Preferably, the protein is released from its binding to the immobilized ligand by the addition of a chemical compound, processing with a reducing agent, processing with a protease, or a change in pH. Preferably, the chemical compound is imidazole, the reducing agent is reduced glutathione, the protease is selected from the list comprising: Pre-Scission, Enterokinase, Thrombin, Tev, HRV 3C, ULP1 and factor Xa. More preferably, the protease is Pre-Scission. Also more preferably, the protease is Enterokinase.

In the case of proteins with at least one polyhistidine marker, they can bind to immobilized nickel (11) and / or cobalt (11) ligands, and be released by the addition of imidazole, or anti-polyhistidine antibodies bound to protein A or G immobilized and be released by a change of pH.

In the case of proteins with at least one GST marker, they can bind to an immobilized glutathione ligand and be released by the addition of reduced glutathione, or anti-GST antibodies bound to immobilized protein A or G and released by a pH change. .

A third aspect of the invention relates to a kit comprising the nucleotide sequence, gene construct or vector of the first aspect of the invention for protein expression and protein purification. Preferably, the vector, called pTOP, contains the SEO ID NO: 1 nucleotide sequence.

or a bioequivalent variant. More preferably, the pTOP vector is the SEO ID NO: 1 nucleotide sequence.

In a preferred embodiment of the third aspect of the invention, the kit further comprises instructions for carrying out the method of the second aspect of the invention.

In a preferred embodiment of the third aspect of the invention, the kit further comprises a protease. Preferably, the protease is Pre-Scission. Preferably, the protease is Enterokinase.

In a preferred embodiment of the third aspect of the invention, the kit further comprises a pair of direct and reverse reactivation oligonucleotide primers of between 30 and 100 nucleotides, where the direct primer sequence coincides with the SEO ID NO: 1 fragment whose end 3 'is the nucleotide at position 1,093 of SEO ID NO: 1 or the nucleotide at a position of up to 10 nucleotides above, and where the reverse primer sequence is complementary to SEO ID NO: 1 so that its 3' end is the nucleotide complementary to the nucleotide at position 1,094 of SEO ID NO: 1

or the nucleotide at a position of up to 10 nucleotides posterior.

The direct and reverse primers could be SEO ID NO: 20 and SEO ID NO: 21, respectively.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Figure 1. Shows the construction process of the pTOP vector. Scheme of the steps for obtaining pTOP that only shows the coding zone with the specific markers and protease targets, the rest of the nucleotide sequence remains unchanged throughout the process. MD: directed mutagenesis using the Site-directed mutagenesis Kit (Stratagene). R: classical cloning by restriction. CiPCR: cloning by reverse cloning PCR. ATG: initiation codon (a); 10H: polyhistidine marker coding sequence (b) and (e); GST: Glutathione-S-transferase marker coding sequence (e); PS: target sequence recognition and cut by the PreScission protease (d); EK: target recognition sequence and cut by the protease Enterokinase (f); ** --_ ##: insertion site of the sequence of interest to be cloned; T AA: translation termination codon (g).

Figure 2. Shows the map of the circular pTOP vector. The insertion site of the sequence of interest to be cloned and a detail of the markers and fusion elements to the sequence of the gene of interest are shown, as well as their order.

Figure 3. Shows the three possible proteins that can be obtained using pTOP for expression and purification. A: Protein of interest fused to the three markers and the two recognition targets for specific proteases. B: Protein of interest fused to a marker and to the recognition target for the Enterokinase protease. C: Protein of interest without extra amino acids.

Figure 4. Shows the obtaining of the PCBP1 protein. A. Westem blot showing the development of arabinose induction of PCBP1 expression in the E. coli BL21 culture containing the pTOPPCBP1 gene construct. The optimal expression conditions of PCBP1 are obtained at 3 hours induction with arabinose. B. Obtaining the recombinant protein PCBP1 fused to the corresponding markers, pure in solution or immobilized in glutathione or Ni balls. Lane 1: 10His-GST-10His-PCBP1 in solution, lane M: molecular weight marker, lane 2 10His-PCBP1 in solution, lane 3: 10His-GST-10His-PCBP1 immobilized in glutathione balls.

EXAMPLES

The invention will now be illustrated by several examples describing the steps for the construction of pTOP and tests carried out by the inventors, which show the specificity and effectiveness of the expression vector of the invention pTOP and the method associated with said vector.

EXAMPLE 1: Construction of the expression vector pTOP.

The pTOP vector was constructed in four stages (Figure 1) from plasmid pPreTOP1-Tfam (SEQ ID NO: 2), where the Tfam gene cloned in pPreTOP1 goes from nucleotide 365 to nucleotide 973 of SEQ ID NO: 2.

Step 1. Change of the BamHI restriction enzyme recognition sequence at position 359-364 of plasmid pPreTOP1-Tfam (SEQ ID NO: 2) to that of restriction enzyme Bgl 11 to obtain plasmid pPreTOP2-Tfam (SEQ ID NO: 3).

It was carried out by PCR-directed mutagenesis in a total volume of 50 ¡JI using the QuikChange® Site-Oirected Mutagenesis Kit (Stratagene). The cold reaction mixture was prepared following the supplier's instructions by adding 5 JI of 10X buffer, 1 JI of dNTPs (deoxyribonucleotide triphosphate), 125 ng of each of the primers, 2.5 U of the polymerase of Pfu Turbo DNA, 69 ng of plasmid pPreTOP1-Tfam and 37 JI of sterile double-distilled water to complete the total 50 JI of the reaction volume.

The PTC-200 thermocycler program (MJ Research) consisted of 1 cycle at 95 oC for 30 seconds, followed by 12 cycles composed of three stages, at 95 oC for 30 seconds, at 55 oC for 1 minute, and 68 oC for 5 minutes After cooling on ice for 2 minutes, it was treated with 2 U of Dpnl for 2 hours at 37 oC. Next, 2 JI of the reaction mixture was used to transform 50 JI of competent E. coli TOP10 cells (provided with the kit) by thermal shock. After 1 hour in orbital shaking at 200 rpm and 37 oC in an incubator-shaker, they were seeded on an LB-agar plate with ampicillin (100 µg / ml).

After 24 hours of incubation at 37 ° C, two of the colonies were inoculated in a 5 ml culture of LB medium with ampicillin (100 µg / ml) and kept at 37 ° C and 200 rpm for 16 hours. After cooling in an ice bath, plasmids were extracted and purified, which were eluted in 50 JI of sterile double-distilled water and visualized on a 1% agarose gel and staining with ethidium bromide (0.5 Jg / ml) Subsequently, two 10 µL total volume reactions were prepared with each of the plasmids, respectively, for cutting with the restriction enzyme Bgl II (Roche), according to the supplier's instructions. It was maintained at 37 oC in a thermoblock for 1 hour and after deactivation of the enzyme for 20 minutes at 65 oC in a thermoblock, the products were analyzed in a 1% agarose gel in T AE buffer containing 0.5 µg / ml of ethidium bromide. 100 bp DNA Ladder (GeneCraft) was used as molecular weight marker. The products were visualized and photographed in the Bio-Vision gels documentation system with Vision-CAPT software. Finally, one of the two plasmids was sequenced with the Big Oye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the electroferograms were obtained in the sequencing service of the University of Santiago de Compostela with the Applied 3730xl O NA Analyzer Biosystems TM. The electropherograms were visualized with the Chromas Lite program (Copyright © 2005 Technelysium Pty Ud), which confirmed the obtaining of plasmid pPreTOP2-Tfam.

Step 2. Simultaneous introduction of the Pre-Scission protease recognition sequence and the Nhel restriction enzyme recognition sequence into plasmid pPreTOP2-Tfam (SEQ ID NO: 3) to obtain plasmid pPreTOP3-Tfam (SEQ ID NO 6).

The cloning of the insert into the vector was carried out by the restriction method by cutting with the restriction enzymes Bgl II (Roche) and Pvull (New England Biolabs) and binding with the T4 ONA ligase (Promega).

Obtaining the insert

Primers were first designed properly to carry out the amplification of the insert by PCR. The direct primer (SEO ID NO: 4), 5'-ttttagatct-ctggaagttctgttccaggggccc-gctagc-ttttccagcatgggtagcta-3 'was provided with an extra sequence at the 5' end containing the target sequence for the restriction enzyme Bgl 11, between positions 5-10 of SEO ID NO: 4, followed by the target sequence of the Pre-Scission protease, between positions 11-34 of SEO ID NO: 4, and the target sequence for the restriction enzyme Nhel, between positions 35-40 of SEO ID NO: 4, and at its 3 'end the sequence for hybridization with the AON to be amplified was included. The reverse primer sequence (SEO ID NO: 5), 5'-catacccat-cagctg-acttggagttag-3 'completely hybridizes with the corresponding template sequence, which already contained the sequence for the Pvull restriction enzyme, and which occupies SEO positions 10-15 NO: 5.

For a total volume of 80! JI of reaction mixture 16 were added cold

! 5x PrimeSTAR buffer JI, direct and reverse primer 0.2! JM each, mix of 0.2 mM dNTPs, 200 ng of the pPreTOP2-Tfam mold (SEQ ID NO: 3), 0.8! JI of PrimeSTARTM HS ONA (Takara) polymerase and sterile double-distilled water to complete total volume.

The amplification program in a PTC-200 thermal cycler consisted of 1 cycle at 95 oC for 5 minutes, followed by 30 cycles composed of three stages, at 95 oC for 30 seconds, at 60 oC for 30 seconds and 72 oC for 1 minute, and a final cycle at 72 oC for 10 minutes. The products were then analyzed on a 1% agarose gel.

After purification by cutting and extracting the band of a preparative agarose gel using the High Pure PCR Product Purification kit (Rache), and sterile double-distilled water elution, the amplified fragment was quantified by spectrophotometry in the Nanodrop 2000C. Two! Jg of the pure amplicon was cold mixed with 4! JI of 10x buffer, 0.4! JI of BSA 100x and 0.5! JI of each restriction enzyme Bgl II (NEB) and Pvull (NEB) sterile double-distilled water up to 40! JI total volume, and incubated at 37 oC overnight in a thermoblock. The products were analyzed and purified again by the same procedure, quantified and stored at -20 oC.

Obtaining the linear vector

Three! Jg of the pure circular pPreTOP2-Tfam plasmid (SEQ ID NO: 3) was cold mixed with 4! JI of 10x buffer, 0.4! JI of BSA 100x and 0.5! JI of each Bglll restriction enzyme ( NEB) And Pvull (NEB) sterile double-distilled water up to 40! JI total volume and incubated at 37 oC overnight. The products were extracted from the band of a preparative agarose gel, purified following the same protocol as for the purification of the insert and after quantification they were stored at -20 ° C.

Insert Cloning

Three hundred ng of the cut insert was mixed with 30 ng of the linear pPrepTOP2-Tfam vector (SEO ID NO: 3), 2 JI of 10x buffer and 1 JI of T4 DNA ligase (NEB) and sterile double-distilled water to complete 20 ¡JI. The mixture was incubated at 16 oC overnight in a thermocycler in a cold chamber. Ninety ¡JI of chemically competent E. coli DH5a bacteria were transformed with 10 ¡JI of the ligation product and seeded on a plate of LB-agar ampicillin (100 ¡Jg / ml). After 24 hours, 23 colonies were analyzed by PCR, of which 21 were positive. The 5 ml liquid culture plasmid of 2 clones was extracted with the GeneElute ™ Plasmid Miniprep kit (Sigma) and after spectrophotometry quantification the presence of the insert was checked by cutting with the Nhel restriction enzyme (NEB) following the instructions of the provider. The correct insertion of the insert into plasmid pPreTOP2-Tfam (SEO ID NO: 3) was carried out by direct sequencing of the plasmid of one of the positive clones, corroborating the obtaining of plasmid pPreTOP3-Tfam (SEO ID NO: 6).

Step 3. Introduction of the Glutathione 5transferase (G5T) coding sequence in plasmid pPreTOP3-Tfam (5EQ ID NO: 6) to obtain plasmid pPreTOP4-Tfam (5EQ ID NO: 9)

The insertion of the GST coding sequence was performed by CiPCR as a cloning method.

Insert Amplification

For the amplification of the GST sequence, the vector pGEX-6P-1 (GE Healthcare) was used as a template. The coding zone for GST extends from position +261 to +917 and, if the coding sequence of the Pre-Scission protease target is included, it covers up to position 941 and adds a total of 680 base pairs, which were amplified.

The PCR was performed in a final volume of 50! JI containing 10! JI of PrimeSTAR 5X buffer, a mixture of 0.2 mM dNTPs each, 0.2! JM of each of the oligonucleotide primers, 50 pg of plasmid pGEX -6P-1 and 1.25 U of PrimeSTARTM HS ONA polymerase (Takara). The oligonucleotide primers were designed with the help of the Primer Express 2.0.0 (Applied Biosystems) program, taking into account the sequence of the pGEX-6p1 mold region from which the insert was amplified (nucleotides SEO SEO 22-35 NO: 7 ) and that of the region of the pPreTOP3-Tfam vector (SEO ID NO: 6) where the insert was cloned (nucleotides 1-21 of SEO ID NO: 7). The direct primer sequence was (SEO ID NO: 7): 5'-catcatcatggcggcagatct-tcccctatactagg-3 'and the reverse sequence (SEO ID NO: 8) 5'-aaagctagc-gggcccctggaacagaacttc -3', which included the coding sequence of the Pre-Scission protease target (nucleotides 1-9 of SEO ID NO: 8). In all reactions, sterile double-distilled water was used as diluent. The amplification conditions in the PTC200 thermal cycler consisted of 1 cycle of denaturation at 94 oC for 30 seconds; 40 cycles composed of three stages, at 98 oC for 10 seconds, at 54 oC for 30 seconds, and at 72 oC for 45 seconds, and 1 final cycle at 72 oC for 10 minutes. The PCR products were electrophoresed on a 1% agarose gel. The correct band was cut with a scalpel blade and the AON fragments were extracted and purified with the High Pure PCR Product Purification Kit (Roche) following the protocol recommended by the supplier, and eluted with sterile double-distilled water. They were then quantified by spectrophotometry in the NanoOrop 2000C and stored at -20 oC until use.

CiPCR for the insertion of the GST coding sequence

The CiPCR was performed in a PTC-200 thermocycler in a final volume of 25! JI containing 5! JI of PrimeSTAR 5X buffer, a mixture of 0.2 mM dNTPs each, 250 ng of the PCR amplicon obtained in the previous section ( whose sequences acted as primers (at the 3 'end), and elongation terminators (at the 5' end) by double hybridization of its two ends with the corresponding sequences of the vector at the selected place to clone the insert), 100 ng of plasmid pPreTOP3-Tfam (SEQ ID NO: 6) and 1.25 U of PrimeSTARTM HS ONA polymerase (Takara). The amplification conditions of the CiPCR were: 1 cycle of denaturation at 95 oC for 5 minutes, 5 cycles composed of four stages, at 95 oC for 1 minute, at 81 ° C for 30 seconds, at 65 oC for 30 seconds, and 72 oC for 6 minutes, 1 four-stage cycle, at 98 oC for 30 seconds, at 81 ° C for 20 seconds, at 65 oC for 20 seconds and 72 oC for 6 minutes. Then, another 10 cycles composed of four stages, at 98 oC for 20 seconds, at 81 ° C for 15 seconds, at 65 oC for 15 seconds and at 72 oC for 6 minutes. Finally, 1 extension cycle at 72 oC for 15 minutes. After cooling on ice, the PCR product was treated with 2 U of the Opnl enzyme (Fermentas) for 6 hours at 37 oC, followed by heating denaturation, and after cooling renaturation the products were precipitated with sodium acetate / ethanol, dried in air, they were diluted in 6 µJ of sterile double-distilled water, which were used to thermally transform 60 µI of chemically competent cells E. coli TG-1. After seeding of the bacteria transformed into LB-agar plates containing ampicillin (100 µg / ml), they were incubated for 24 hours. The colonies obtained were analyzed by PCR, and the PCR products were visualized on 1% agarose gels in TAE buffer. Two of the positive colonies were seeded in liquid LB containing ampicillin (100 µg / ml) and grown for 16 hours. The culture was cooled on ice, the cells were precipitated by centrifugation at 3,000 x g for 20 minutes, the supernatant was decanted and the plasmid was extracted from the GenElute plasmid purification kit (Sigma), following the supplier's instructions. The presence of the insert in the plasmids was reconfirmed by PCR and these were introduced into chemically competent E. coli BL21 cells by heat shock transformation. Two colonies were selected, which were grown in liquid LB culture containing ampicillin (100 µg / ml) for 16 hours. To a 0.5 ml aliquot of the liquid culture of each of the two colonies was added 25% sterile glycerol and both were stored at -80 oC. From the rest of the culture, the two plasmids were extracted again for the analysis and corroboration of the sequences by direct sequencing of the plasmid, which corresponded to those expected for plasmid pPreTOP4Tfam (SEO ID NO: 9).

Step 4. Introduction of the 10 histidine epitope and the recognition sequence for the Enterokinase protease recognition in plasmid pPreTOP4Tfam (SEQ ID NO: 9) to obtain plasmid pTOP-Tfam (SEQ ID NO:

19).

The insertion of the 10 histidine epitope (1 OHis or 1 OH) and the coding sequence of the cutting target with Enterokinase (EK) was carried out directly by CiPCR with the primers: direct primer, (SEO ID NO: 10) 5 ' gaagttctgttccaggggccc-catcaccatcaccatcaccatcaccatcac-gatgacgatgaca ag-ttttccagcatgggtagctatcca-3 ', which contains the coding sequence of the 10 His, between nucleotides 22-51, that of the EK target, between nucleotides 52-64, the sequence of the 5' ends between nucleotides 1-21, it is the termination signal of the polymerase elongation and that of the 3 'end is the hybridization sequence with the pPreTOP4-Tfam template (SEO ID NO: 9); the reverse primer (SEO ID NO: 11) with the sequence 5'aaagctagcgggcccctggaacagaacttc-3 ', fully hybridizes with the coding area of the Pre-Scission protease target of the template and insert. The final volume of the CiPCR mixture was 25! JI and contained 5! JI of 5X buffer, 0.2 mM dNTP mix each, 0.2! JM of each of the primers, 100 ng of plasmid pPreTOP4- Tfam and 1.25 U of PrimeSTARTM HS DNA polymerase (Takara). In all reactions, sterile double-distilled water was used as diluent. The amplification program in the PTC-200 thermal cycler consisted of 1 cycle of denaturation at 95 oC for 5 minutes, 5 amplification cycles composed of three stages, at 95 oC for 1 minute, at 70 oC for 30 seconds, at 72 oC for 7 minutes A cycle composed of three stages, at 98 oC for 30 seconds, at 70 oC for 20 seconds and at 72 oC for 7 minutes. He continued with 10 cycles composed of three stages, at 98 oC for 20 seconds, at 70 oC for 15 seconds and at 72 oC for 7 minutes and, finally, a final cycle at 72 oC for 15 minutes. After cooling on ice, the PCR product was treated with 2 U of Opnl (Fermentas) for 6 hours at 37 oC, followed by precipitation with sodium acetate / ethanol. After centrifugation of the cold samples at maximum speed for 20 minutes, the supernatant was discarded and the precipitate obtained was washed twice with 100 µI of 70% ethanol followed by cold centrifugation at maximum speed for 20 minutes. After complete drying of the precipitate in the air, said precipitate was dissolved in 5 µI of sterile double-distilled water. With the product, 60! JI of chemically competent cells were transformed

E. coli TG-1 by thermal shock. The culture was seeded on an agarchocolate plate (Becton-Oickinson) with ampicillin (200 µg / ml) and incubated at 37 ° C for 24 hours.

The colonies obtained were analyzed by PCR with the primers (SEO ID NO: 12) 5'-ctaggctggaagttctgttccaggggcccg-3 'and inverse (SEO ID NO: 13) 5'ttttgaattc-ctctttatacttgctcacagcttcttt, and the PCR products were analyzed on agarose gels 1% in TAE buffer. The colonies of our interest were the negative ones and these were preselected for a second analysis also by PCR with the primers direct (SEO ID NO: 14) 5'gaccaaagccatgacaaaaacg-3 'and inverse (SEO ID NO: 15) 5'tcgacggcgctattcagatc-3 '. In this case, the amplicons of the positive colonies of the correct size (1,670 base pairs) were cut with Nhel following the supplier's instructions (NEB). The correct plasmids were those that would not be digested with Nhel. Two of the colonies corresponding to amplicons that were not cut with Nhel were seeded in liquid LB and grown for 16 hours at 37 oC with orbital shaking at 200 rpm. The cultures were cooled on ice, the cells were precipitated by centrifugation at 3,000 x 9 for 20 minutes, the supernatant was decanted and the two plasmids contained in the precipitates were extracted with the GenElute ™ Plasmid Miniprep (Sigma) plasmid purification kit following The supplier's instructions. The presence of the insert in two plasmids was again confirmed by PCR, and E. coli BL21 were chemically competent cells introduced by heat shock transformation as before. Two colonies of one of the plates were selected, grown in liquid LB culture containing ampicillin (100 µg / mL), the plasmid was extracted again and 25% glycerol was added to an aliquot of the liquid culture of each colony sterile and stored at -80 oC. The extracted plasmids were purified and sequenced, corroborating the expected sequence for plasmid pTOPTfam (SEO ID NO: 19). In SEO ID NO: 19, the initiation codon comprises nucleotides 320-322, the sequence encoding the first polyhistidine marker comprises nucleotides 326-355, the sequence encoding the second GST marker comprises nucleotides 356-1.024 , the sequence encoding the Pre-Scission protease target comprises nucleotides 1,025-1,048, the sequence encoding the third polyhistidine marker comprises nucleotides 1,049-1,078, and the sequence encoding the Enterokinase protease target comprises nucleotides 1,079-1,093.

EXAMPLE 2: Cloning in pTOP of a recombinant protein by CiPCR: Obtaining recombinant PCB-1 (poly-C-binding protein 1)

Cloning of PCBP1 in pTOP

PCBP1 was amplified from a complementary AON (cONA) obtained by retrotranscription with the M-MLV retrotranscriptase (Invitrogen) from blood lymphocyte messenger RNA extracted by trizol (Invitrogen). The primers for the PCR were the direct primer (SEO ID NO: 16) 5'cacgatgacgatgacaag-gatgccggtgtgactgaa-3 'and the reverse primer (SEO ID NO: 17) 5'-atgacaactccgtcttcc-ctagctgcaccccat-3', designed based on the cONA sequence of the PCBP1-001 ENST00000303577 transcript, whose coding region contains 1,071 base pairs, according to the Ensembl database. The 3 'ends of the oligonucleotide primers (nucleotides 19-36 of SEO ID NO: 16 and nucleotides 19-35 of SEO ID NO: 17) correspond to the sequence complementary to the site of hybridization with PCBP1. The resulting amplicon is from

1,104 base pairs, which corresponds to the PCBP1 cONA sequence from position 294 to 1,362 (1,068 base pairs, excluding the initiation ATG). The sequences of the 5 'ends (nucleotides 1-18 of SEO ID NO: 16 and nucleotides 1-18 of SEO ID NO: 17) and their complemenaries of the 3' ends of the amplicon of 1,104 base pairs, hybridize with the pTOP vector on sites ** --_ ##.

The PCR amplification of PCBP1 (insert) was performed in a final volume of 50 ¡JI containing 10 ¡JI of PrimeSTAR 5X buffer, mixture of 0.2 mM dNTPs each, 0.2 ¡JM of each of the primers , 50 ng of CONA and 1.25 U of PrimeSTAR HS ONA polymerase (Takara) and as diluent sterile double-distilled water. The amplification was carried out in the PTC-200 thermocycler with the program of 1 cycle of denaturation at 94 oC for 30 seconds; 40 cycles composed of three stages, at 98 oC for 10 seconds, at 60 oC for 30 seconds, and 72 oC for 75 seconds and 1 final cycle at 72 oC for 10 minutes, to obtain the amplicon of 1,104 base pairs, which was analyzed on a 1% agarose gel in TAE buffer containing ethidium bromide (0.5 JI / ml in TBE) and comparing it with the 1K AON molecular weight marker of GeneCraft. It was visualized with UV light and photographed in the Bio-Vision gels documentation device. After cutting the band with a scalpel blade and purifying the products with the PCR product purification kit (Rache), they were eluted with sterile double-distilled water and stored at -20 ° C until used in the CiPCR.

In the CiPCR, the sequences of each of the 5 'and 3' ends of each of the chains of the 1,104 base pair amplicons hybridized with each of the two complementary sequences of the sites ** --_ ## corresponding to each chain of the vector where PCBP1 was cloned (SEO ID NO: 19), therefore being those of the 3 'ends (complementary to the corresponding parts of each of the 5' ends of the direct and reverse primer oligonucleotides of the first PCR), those that acted as primers in the CiPCR, and the sequences of the 5 'ends (directly contributed by the oligonucleotide primers to the 1,104 base pair amplicons of the first PCR) as termination signals of the elongation . For the CiPCR, a mixture of 25! JI containing 5! JI of PrimeSTAR 5X buffer, 0.2 mM dNTPs each, 250 ng of the PCBP1 amplicon, 100 ng of plasmid pTOP-Tfam and 1.25 U was prepared. of PrimeSTAR HS DNA polymerase (Takara). The amplification conditions in the PTC200 thermal cycler were 1 cycle of denaturation at 95 oC for 5 minutes, 5 cycles composed of four stages, at 95 oC for 1 minute, at 65 oC for 30 seconds, at 52 oC for 30 seconds, and 72 oC for 7.5 minutes, 1 cycle composed of four stages, at 98 oC for 30 seconds, at 65 oC for 20 seconds, at 52 oC for 20 seconds and 72 oC for 7.5 minutes, followed by 10 cycles composed of three stages, at 98 oC for 20 seconds, at 80 oC for 15 seconds and at 72 oC for 7.5 minutes; and 1 final cycle at 72 oC for 15 minutes. The CiPCR products were treated with 2 units of Dpnl for 6 hours at 37 oC. After deactivating the enzyme for 20 minutes at 80 ° C, the products were precipitated with sodium acetate / ethanol, resuspended in 5 µI of sterile double-distilled water, and transformed into E. coli TG1 for analysis and selection of positive clones by PCR Then, two of the positive clones were grown in 5 ml of LB liquid medium with ampicillin (100 µg / ml) and plasmids were extracted. The presence of the insert was checked again by PCR and an aliquot of each was transformed into E. coli strain BL21. A culture of 5 ml of each was obtained, of which 0.5 ml was used to prepare the glycerol strains and store them at -80 oC as a source of bacteria for future cultures. From the rest of the culture volume the plasmids were extracted again, the presence of the insert was checked by PCR and one of the plasmids pTOP-PCBP1 (SEQ ID NO: 18) was sequenced to corroborate that the insertion site and the sequence of the gene PCBP1 and the rest of the coding regions of the labels and targets were correct.

EXAMPLE 3: Optimization of the conditions of induction of plasmid pTOP-PCBP1 and obtaining and purification of recombinant PCBP1 fused with different elements:

3.1-Fused with the two polyhistidine and Glutathione 5Transferase labels (10His-G5T-10His-PCBP1) immobilized or in solution

3.2-Cut with the Pre-5cission protease to obtain PCBP1 fused with the 10His label (10His-PCBP1)

Optimization of the conditions for induction of PCBP1 expression in plasmid pTOP

For the optimization of the conditions of induction of the expression of PCBP1 in the plasmid pTOP, the bacterial reserve strains E. coli BL21 were used with the plasmid pTOP-PCBP1 (SEQ ID NO: 18) that were stored in LB-glycerol a -80 oC. An aliquot was seeded on a plate of LB-agar ampicillin (100 µg / ml). One of the plaque colonies was used fresh to inoculate 5 ml of LB in a 25 ml capacity tube that was grown for 6 hours at 37 oC with stirring. This fresh culture was used to inoculate a flask containing 100 ml of LB with ampicillin (100 µg / ml). It was grown until it reached an approximate density of 0.4 units of optical density, measured in a Biomate3 spectrophotometer (Thermo Scientific) at a wavelength of 600 nm. Next, the expression of PCBP1 was induced by the addition of 0.2% arabinose (from a 20% sterile solution in double-distilled water). 1 ml aliquots of the culture were collected every hour in plastic cuvettes for 1 ml spectrophotometry, until after 6 hours of induction. Their optical densities were measured as they were collected and then the contents were transferred to the corresponding tubes, which were cooled in an ice bath, centrifuged at maximum speed for 1 minute and the precipitates were stored at -80 ° C. . Once the process was finished, the precipitates were transferred from the freezer to an ice bath and resuspended in the lysis buffer volume (50 mM Tris-CIH pH 7.9, 0.5M NaCI, 1 mM leupeptin, 1 mM PMSF and 1 mM corresponding aprotinin), so that all carry the same concentration of bacteria as the sample at induction time. These cell suspensions were then sonicated in the Ultrasonic Homogenizer ultrasonic apparatus at 50% capacity for 4 seconds 4 times with 5-second cooling on ice and then subjected to three stages of freezing (rapid dry ice) and thawing in ice. After cold centrifugation at 15,000 xg for 20 minutes, the supernatants were separated from the precipitates and, after incubation of an equal aliquot of each of the supernatants with loading buffer at 95 ° C for 2 minutes, the aliquots were deposited in 12% SDS-PAGE polyacrylamide mini-wells (37.5: 1 acrylamide: bisacrylamide, Bio-Rad) 0.75 mm thick, 9 cm wide and 7 cm high (Mini-PROTEAN 3, Bio- Rad) After the separation of the protein bands by electrophoresis, they were transferred to PVDF membranes previously activated with methanol. The transfer was carried out in solution with the Mini Trans-Blot® transfer equipment (Bio-Rad) for 2 hours at 60 v cold and then the protein was analyzed by Westem blot. For this, the membranes were washed with PBS containing 0.05% Tween-20 (Sigma) and left blocking overnight at 4 ° C in PBS containing 0.05% Tween-20 and 10% skim milk in powder. The next day they were washed 3 times with PBS containing 0.05% Tween-20 and incubated with 5 ml of a 1: 2,500 dilution of the anti-GST mouse monoclonal antibody (Santa Cruz Biotechnology) for 1 hour at room temperature. Then, the membrane was washed 3 times with PBS containing 0.05% Tween-20 and the membrane was incubated with 5 ml of a 1: 5,000 dilution of the radiotherapy peroxidase conjugated secondary anti-mouse antibody (HRP) (Amersham ) for 1 hour in slow orbital agitation at room temperature. After 3 washes with PBS containing 0.05% Tween-20, the membranes were developed by a chemiluminescence assay. For this, a Lumigen ™ PS-3 solution (Amersham) was used as a substrate, following the recommendations of the supplier. The membrane was incubated for 1 minute with the substrate and then made to the consecutive exposure of several radiographic films (Hyperfilm ECL, Amersham) in the dark, the first for 1 minute in the Curix Compact Plus automatic device, Agfa, which after development allowed to make an approximate calculation of the exposure time necessary to obtain the signal with the satisfactory intensity. The following films were exposed immediately after the calculation of the necessary exposure time. The result is shown in the Westem blot of Figure 4A.

Next, the process was scaled to obtain PCBP1 in the quantities required for each type of test. 1 liter of LB containing 100 µg / ml of ampicillin was inoculated with 50 ml of a previous culture inoculated with the corresponding bacteria from the glycerol stock pool at -80 oC previously seeded on a plate of LB-ampicillin agar (100¡ Jg / ml). This culture was incubated at 37 oC and 200 rpm until reaching a turbidity of 0.4 units of optical density at 600 nm wavelength. 0.2% arabinose was added and maintained in culture for 3 hours (as deduced from the commissioning experiment, see Figure 4A). Then, the culture was cooled on ice, transferred to the corresponding bottles and centrifuged at 3,000 x g for 20 minutes in the cold. The precipitates were resuspended in 10 ml of lysis buffer and sonicated in an Ultrasonic Homogenizer at 50% capacity for 10 seconds, 10 times with 30 second cooling intervals on ice. After three freeze-thaw cycles on dry ice and ice, it was cold centrifuged at 15,000 x g for 20 minutes. The supernatant was incubated with 200 JI of Ni-NTA balls (Invitrogen), previously washed 3 times with 1 ml of PBS for 2 hours in cold and slow orbital agitation. After centrifugation and decanting of the supernatant, two washes of the precipitate were made with lysis buffer containing 1% triton X-100 (Sigma) by incubation for 5 minutes with slow orbital agitation in cold, another two with lysis buffer, and another two with lysis buffer containing 50 mM imidazole. Centrifugations between washings were done cold at 2,000 x g for 2 minutes. Then, 200 µI of lysis buffer containing 1 M of imidazole was added, stirred for 5 minutes in the cold and slow rotational stirring for elution of the balls. After a new centrifugation, the supernatant was transferred to another tube and the process was repeated a second time. Once the two volumes of supernatant containing the eluted protein were combined, a cold aliquot of 10! JI (containing 1 OHis-GST1 OHis-PCBP1, Figure 4B lane 1) was stored, and the rest were added cold PBS until complete 1mi and 150! JI of immobilized glutathione in balls (Amersham Biosciences), previously washed 3 times with 1 ml of cold PBS. After cold incubation with slow orbital agitation for 2 hours, two washes were performed with lysis buffer containing 1% triton X-100, two others with lysis buffer and two others with Pre-Scission protease (Amersham) protease buffer. ), all by cold incubation and slow rotational agitation for 5 minutes and cold centrifugation at 2,000 xg for 2 minutes. After the last wash, 70! JI of PreScission protease cut buffer was added to the precipitate of the balls, a 2! JI aliquot was stored (containing 1 OHis-GST-1 OHis-PCBP1 attached to the glutathione balls , Figure 4B lane 3). Half was stored for use in assays that required the protein immobilized in the balls and the other half was added with 35! JI of protease cutting buffer and 4! JI of Pre-Scission protease and the mixture was incubated with rotary agitation Slow for 48 hours collecting aliquots of 65! JI every 12 hours with replacement of new cutting buffer (the PreScission protease has a GST tail so it remains attached to the balls and does not need to be replaced). 25% glycerol was added to each aliquot and stored at -80 ° C until use (these aliquots contain 1 OHis-PCBP1). The protein immobilized in the balls was stored as such at 4 oC for immediate use. The result can be seen in Figure 4B.

Claims (51)

1.-A nucleotide sequence comprising the following elements:

to.

an initiation codon,

b.

a nucleotide sequence encoding an M marker,

C.

a nucleotide sequence encoding an N marker,

d.

a nucleotide sequence encoding a protease recognition amino acid sequence P,

and.

a nucleotide sequence encoding a 0 marker,

F.

a nucleotide sequence encoding a protease recognition amino acid sequence Q,

g.

a termination codon.

2. The nucleotide sequence according to the preceding claim comprising a sequence consisting essentially of the following elements:

to.

an initiation codon,

b.

a nucleotide sequence encoding an M marker,

C.

a nucleotide sequence encoding an N marker,

d.

a nucleotide sequence encoding a protease recognition amino acid sequence P,

and.

a nucleotide sequence encoding a 0 marker,

F.

a nucleotide sequence encoding a protease recognition amino acid sequence Q,

g.

a termination codon.

3. The nucleotide sequence according to any of the preceding claims comprising the elements in the following order: an initiation codon, a nucleotide sequence encoding an M marker, a nucleotide sequence encoding an N marker, a nucleotide sequence that encodes for an amino acid sequence P of recognition of a protease, a nucleotide sequence that encodes for an O marker, a nucleotide sequence that encodes for an amino acid sequence Q of recognition of a protease, a termination codon. 4. The nucleotide sequence according to any of the preceding claims characterized in that the M and O markers are the same. 5. The nucleotide sequence according to any of the preceding claims characterized in that the M, NYO markers are selected from the list comprising: polyhistidine, Glutathione S-transferase, avidin, streptavidin, hemagglutinin, VSV-G, HSVtk, FLAG, protein binding to maltose, V5, myc and a fluorescent protein. 6. The nucleotide sequence according to the preceding claim wherein the markers M, N and O are selected from polyhistidine and Glutathione Stransferase. 7. The nucleotide sequence according to the preceding claim wherein the M and O markers are a polyhistidine and the N marker is Glutathione Stransferase. 8. The nucleotide sequence according to the preceding claim wherein the M and O markers are a polyhistidine of between 6 and 14 histidines, preferably 10 histidines. 9. The nucleotide sequence according to any of the preceding claims characterized in that the amino acid sequences P and Q of protease recognition are selected from the list comprising the protease targets: Pre-Scission, Enterokinase, Thrombin, Tev, HRV 3C, ULP1 and factor Xa. 10. The nucleotide sequence according to the preceding claim wherein the amino acid sequence P is the recognition target of the PreScission protease and the amino acid sequence Q is the recognition target of the Enterokinase protease. 11. The nucleotide sequence according to any of the preceding claims which further comprises an inducible promoter. 12. The nucleotide sequence according to the preceding claim wherein the inducible promoter is selected from the list comprising: arabinose inducible promoter, lactose inducible promoter or IPTG (isopropyl- ~ -thiogalactoside), ecdysone inducible promoter, tetracycline inducible promoter , galactose inducible promoter, heavy metal inducible promoter, mifepristone inducible promoter, tryptophan inducible promoter, temperature inducible promoter, phosphate inducible promoter and methanol inducible promoter. 13.-The nucleotide sequence according to the preceding claim wherein the promoter is arabinose inducible. 14. The nucleotide sequence according to any of the preceding claims which further comprises at least one origin of replication. 15. The nucleotide sequence according to the preceding claim comprising the origin of replication of plasmid pUCo 16. The nucleotide sequence according to any of the preceding claims which further comprises at least one selection gene. 17. The nucleotide sequence according to the preceding claim wherein the selection gene is chosen from the list comprising: an antibiotic resistance gene, a gene that codes for a fluorescent protein, a gene that codes for a luminescent protein, a gene which codes for betagalactosidase and a gene that converts auxotrophic strains into prototrophs. 18.-The nucleotide sequence according to the preceding claim wherein the selection gene is an antibiotic resistance gene. 19. The nucleotide sequence according to the preceding claim wherein the selection gene is a gene for resistance to ampicillin, kanamycin, tetracycline, chloramphenicol, geneticin, hygromycin B, puromycin, blasticidine, methotrexate or zeocin. 20.-The nucleotide sequence according to the preceding claim wherein the selection gene is an ampicillin resistance gene. 21. The nucleotide sequence according to any of the preceding claims characterized in that it is SEQ ID NO: 1 or a bioequivalent variant. 22. The nucleotide sequence according to any of the preceding claims characterized in that it is a gene construct or is part of a gene construct. 23.-The nucleotide sequence according to the preceding claim characterized in that it is a gene construct. 24. The nucleotide sequence according to any of the two preceding claims characterized in that it is a vector or is part of a vector. 25. The nucleotide sequence according to the preceding claim characterized in that it is a vector. 26.-A method of gene expression and protein purification characterized in that it comprises a step (h): h. Cloning a gene of interest in a nucleotide sequence, gene construct or vector according to any one of claims 1 to 25. 27. The method according to the preceding claim characterized in that after step (h) the following stage (i) is carried out: i. Produce the protein encoded by the sequence cloned in step (h). 28. The method according to the preceding claim characterized in that step (i) comprises at least two sub-stages: i '. Enter the nucleotide sequence, gene construct or vector obtained in step (h) in a prokaryotic cell or in a cell eukaryotic, i ". Cultivate the cell of stage (i ') in the presence of substances necessary for the protein encoded by the gene to be expressed cloned interest in stage (h). 29. The method according to the preceding claim wherein in step (i ') the nucleotide sequence, gene construct or vector is introduced into a prokaryotic cell, preferably Eseheriehia eoli. 30. The method according to claim 28 characterized in that in step (i ') the nucleotide sequence, gene construct or vector is introduced into a yeast cell, an insect cell or a mammalian cell. 31. The method according to any of the three preceding claims wherein the cultivation of step (i ") is done in the presence of arabinose. 32. The method according to any of the four preceding claims, which further comprises the following step (j): j. Purify the protein produced in step (i). 33. The method according to the preceding claim characterized in that step U) comprises at least three sub-stages: j '. List the cultured cells in step (i "). j ". Bind the lysate protein obtained in step (j ') to a ligand immobilized on a solid support. j "'. Release the protein from its binding to the U-stage ligand"). 34. The method according to the preceding claim wherein the ligand immobilized on a solid support of step U ") is Glutathione. 35. The method according to claim 33 wherein the ligand immobilized on a solid support of step (j ") is a metal ion. 36. The method according to the preceding claim wherein the metal ion is nickel. (11) and / or cobalt (11). 37. The method according to any of the four preceding claims wherein the protein is released from its binding to the ligand by at least one of the following forms: a change in pH, the addition of free ligand, the addition of a molecule that moves to the protein by competition for ligand binding, treatment with a reducing agent, processing with a protease. 38. The method according to the preceding claim wherein the protein is released from its binding to the ligand by processing with a protease. 39. The method according to any of the two preceding claims wherein the protease is selected from the list comprising: Pre-Scission, Enterokinase, Thrombin, Tev, HRV 3C, ULP1 and factor Xa. 40. The method according to the preceding claim comprising the PreScission protease. 41. The method according to claim 39 comprising the protease Enterokinase. 42. The method according to claim 37 wherein the molecule that competes for ligand binding is imidazole. 43. The method according to claim 37 wherein the reducing agent is reduced glutathione. 44.-A kit comprising a nucleotide sequence, a gene construct or a vector according to any one of claims 1 to 25 for protein expression and purification. 45. The kit according to the preceding claim wherein the vector is the nucleotide sequence SEQ ID NO: 1 or a bioequivalent variant. 46. The kit according to any of the two preceding claims wherein the vector is the nucleotide sequence SEQ ID NO: 1. 47. The kit according to any of the three preceding claims which further comprises the instructions for carrying out the methods according to any of claims 26 to 43. 48. The kit according to any of the four preceding claims which further comprises at least one protease. 49. The kit according to the preceding claim wherein the protease is Pre-Scission. 50. The kit according to claim 48 wherein the protease is Enterokinase. 51. The kit according to any of the previous seven claims comprising a pair of direct and reverse primers between 30 and 100 nucleotides, where the sequence of the direct primer coincides with the SEO ID NO: 1 fragment and whose 3 'end is the nucleotide at position 1.093 of SEO ID NO: 1, and where the reverse primer sequence is complementary to SEO ID NO: 1 so that its 3 'end is the nucleotide complementary to the nucleotide at position 1.094 of SEO ID NO: one.  FIG. 1 BarnHI ATG  lOHEg, Wml:  J, ....-- Bglll MD BarnHI ~~ BgllI  I ATG IlOHEg, RW: : ······· '····' ··················· 1 ::::: ~ § ::: t R Nhel Bglll Nhel IA;, .., ;;.;. T ... ;; ... G ~: t :: ;;:;: QH ~. ::::::::: I; · .... .. ~ i ........._ Il! I: CiPCR r- BgllI Nhel CiPCR  B ~ t¡ "~ .., mllllH1  pPreTOPl  4731 bp  pPreTOP2  4734 bp  pPreTOP3  4764 bp  pPreTOP4  5421 bp  pTOP  5460 bp FIG. 2  pTOP 4854 bp FIG. 3 Protein  FIG. 4 TO or 1 2 3 4 S ti B gives 58 48 36, 526 - SEQUENCE LIST <110> University of Santiago de Compostela <120> pTOP: New protein expression and purification vector <130> 1596.30 <160> 23 <170> PatentIn version 3.5 <210> 1 <211> 4854 <212> DNA <213> Artifi ci al Sequence <220> <223> Vector pTOP <400> 1 aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct 60 tctcgctaac caaaccggta accccgctta ttaaaagcat agcgggacca tctgtaacaa 120 aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg 180 attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg 240 atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg tttttttggg 300 ctaacaggag gaattaacca tgggccacca ccatcaccac caccatcatc atcatggcgg 360 cagatcttcc cctatactag gttattggaa aattaagggc cttgtgcaac ccactcgact 420 tcttttggaa tatcttgaag aaaaatatga agagcatttg tatgagcgcg atgaaggtga 480 taaatggcga aacaaaaagt ttgaattggg tttggagttt cccaatcttc cttattatat 540 tgatggtgat gttaaattaa ggccatcata cacagtctat cgttatatag ctgacaagca 600 caacatgttg ggtggttgtc caaaagagcg tgcagagatt tcaatgcttg aaggagcggt 660 tttggatatt agatacggtg tttcgagaat tgcatatagt aaagactttg aaactctcaa 720 agttgatttt cttagcaagc tacctgaaat gctgaaaatg ttcgaagatc gtttatgtca 780 taaaacatat ttaaatggtg atcatgtaac ccatcctgac ttcatgttgt atgacgctct 840 tgatgttgtt tt atacatgg acccaatgtg cctggatgcg ttcccaaaat tagtttgttt 900 taaaaaacgt attgaagcta tcccacaaat tgataagtac ttgaaatcca gcaagtatat 960 agcatggcct ttgcagggct ggcaagccac gtttggtggt ggcgaccatc ctccaaaatc 1020 ggatctggaa gttctgttcc aggggcccca tcaccatcac catcaccatc accatcacga 1080 tgacgatgac aagtaaggaa gacggagttg tcattggaat tcgaagcttg ggcccgaaca 1140 aaaactcatc tcagaagagg atctgaatag cgccgtcgac catcatcatc atcatcattg 1200 agtttaaacg gtctccagct tggctgtttt ggcggatgag agaagatttt cagcctgata 1260 cagattaaat cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc 1320 gcggtggtcc cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt 1380 agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc 1440 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag 1500 taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg 1560 ccgccataaa ggcaggacgc ctgccaggca tcaaattaag tcctgacgga cagaaggcca 1620 tggccttttt gcgtttctac aaactctttt tgtttatttt tctaaataca ttcaaatatg 1680 tgagacaata tatccgctca accctgataa atgcttcaat aatattgaaa aaggaagagt 1740 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 1800 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg gttgggtgca ctgaagatca 1860 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 1920 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 1980 cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 2040 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 2100 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 2160 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 2220 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 2280 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 2340 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 2400 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 2460 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 2520 acgacgggga gtcaggcaac tatgg ATGAA cgaaatagac agatcgctga gataggtgcc 2580 agcattggta tcactgatta actgtcagac caagtttact catatatact ttagattgat 2640 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 2700 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 2760 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 2820 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 2880 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta 2940 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 3000 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 3060 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 3120 cctacaccga gagcgaacga actgagatac ctacagcgtg agctatgaga aagcgccacg 3180 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 3240 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 3300 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 3360 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 3420 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 3480 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 3540 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg 3600 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 3660 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 3720 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 3780 gaggttttca gcatgtgtca ccgtcatcac cgaaacgcgc gaggcagcag atcaattcgc 3840 gcgcgaaggc gaagcggcat gcataatgtg cctgtcaaat ggacgaagca gggattctgc 3900 aaaccctatg ctactccgtc aagccgtcaa ttgtctgatt cgttaccaat tatgacaact 3960 tgacggctac atcattcact ttttcttcac aaccggcacg gaactcgctc gggctggccc 4020 cggtgcattt tttaaatacc cgcgagaaat agagttgatc gtcaaaacca acattgcgac 4080 cgacggtggc gataggcatc cgggtggtgc tcaaaagcag cttcgcctgg ctgatacgtt 4140 ggtcctcgcg ccagcttaag acgctaatcc ctaactgctg gcggaaaaga tgtgacagac 4200 gcgacggcga caagcaaaca tgctgtgcga cgctgg CGAT atcaaaattg ctgtctgcca 4260 ggtgatcgct gatgtactga caagcctcgc gtacccgatt atccatcggt ggatggagcg 4320 actcgttaat cgcttccatg cgccgcagta acaattgctc aagcagattt atcgccagca 4380 gctccgaata gcgcccttcc ccttgcccgg cgttaatgat ttgcccaaac aggtcgctga 4440 gtgcgcttca aatgcggctg tccgggcgaa agaaccccgt attggcaaat attgacggcc 4500 agttaagcca ttcatgccag taggcgcgcg gacgaaagta aacccactgg tgataccatt 4560 cggatgacga cgcgagcctc ccgtagtgat gaatctctcc tggcgggaac agcaaaatat 4620 cacccggtcg gcaaacaaat tctcgtccct gatttttcac caccccctga ccgcgaatgg 4680 tgagattgag aatataacct ttcattccca gcggtcggtc gataaaaaaa tcgagataac 4740 cgttggcctc aatcggcgtt aaacccgcca ccagatgggc attaaacgag tatcccggca 4800 gcagggcccccccccacccccc tcctc tcgtctc ctctc tcgctc tcctc tctctc tctc tctctc tctctc tctctc tctctc tctctc tctctc tctctc tctctc tctctc tctctc tctctc tctctc tctc tctc tctctcc <210> 2 <211> 4731 <212> DNA <213> Artifi ci al Sequence <220> <223> Sequence of plasmid ppreTOPI-Tfam <400> 2 aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct 60 tctcgctaac caaaccggta accccgctta ttaaaagcat agcgggacca tctgtaacaa 120 aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg 180 attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg 240 atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg tttttttggg 300 ctaacaggag gaattaacca tgggccacca ccatcaccac caccatcatc atcatggcgg 360 atccttttcc agcatgggta gctatccaaa gaaacctatg agttcatacc ttcgattttc 420 cacagaacag ctacccaaat ttaaagctaa acacccagat gcaaaacttt cagaattggt 480 taggaaaatt gcagccctgt ggagggagct accagaagca gaaaaaaagg tttatgaagc 540 gctgagtgga tgattttaaa aagcatacaa agaagctgtg agcaagtata aagagcagct 600 aactccaagt cagctgatgg gtatggagaa ggaggcccgg cagagacggt taaaaaagaa 660 agcactggta aagagaagag aattaatttt gcttggaaaa ccaaaaagac ctcgttcagc 720 atataacatt tatgtatctg aaagcttcca ggaggcaaag gatgattcgg ctcagggaaa 780 attgaagctt gtaaatgagg cttggaaaaa tctgtctcct gaggaaaagc aggcatatat 840 aaagatgata tcagcttgct cgacaatgaa ggattcgtta gggaagagca atgaagtctt 900 gttggacgaa gatggctgaa gtgatctcat ccgtcgaagt gtgaaacgat ccggagacat 960 ctctgagcat taaggaagac ggagttgtca ttggaattcg aagcttgggc ccgaacaaaa 1020 actcatctca gaagaggatc tgaatagcgc cgtcgaccat catcatcatc atcattgagt 1080 ttaaacggtc tccagcttgg ctgttttggc ggatgagaga agattttcag cctgatacag 1140 attaaatcag aacgcagaag cggtctgata aaacagaatt tgcctggcgg cagtagcgcg 1200 gtggtcccac CTGAC CCCat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt 1260 gtggggtctc cccatgcgag agtagggaac tgccaggcat caaataaaac gaaaggctca 1320 gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag 1380 gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc 1440 aggacgcccg ccataaactg ccaggcatca aattaagcag aaggccatcc tgacggatgg 1500 cctttttgcg tttctacaaa ctctttttgt ttatttttct aaatacattc aaatatgtat 1560 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg 1620 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt 1680 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga 1740 tcgaactgga gtgggttaca tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa 1800 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt 1860 gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt 1920 cagtcacaga gagtactcac aaagcatctt acggatggca tgacagtaag agaattatgc 1980 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga 2040 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat 2100 cggagctgaa cgttgggaac tgaagccata ccaaacgacg agcgtgacac cacgatgcct 2160 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc 2220 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 2280 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 2340 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 2400 acggggagtc aggcaactat aatagacaga ggatgaacga aggtgcctca tcgctgagat 2460 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta 2520 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc 2580 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa 2640 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca 2700 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta 2760 gcagagcgca actggcttca gataccaaat actgtccttc tagtgtagcc gtagttaggc 2820 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca 2880 gtggctgctg ccagtggcga taagtc gtgt cttaccgggt tggactcaag acgatagtta 2940 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag 3000 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt 3060 gaaaggcgga cccgaaggga caggtatccg gtaagcggca gggtcggaac aggagagcgc 3120 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac 3180 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac 3240 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc 3300 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat 3360 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag 3420 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc 3480 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc 3540 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac 3600 gctcccggca gggcttgtct tccgcttaca gacaagctgt gggagctgca gaccgtctcc 3660 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagcagatc aattcgcgcg 3720 gcggcatgca cgaaggcgaa taatgtgcct g tcaaatgga attctgcaaa cgaagcaggg 3780 ccctatgcta ctccgtcaag ccgtcaattg tctgattcgt taccaattat gacaacttga 3840 cggctacatc attcactttt tcttcacaac cggcacggaa ctcgctcggg ctggccccgg 3900 tgcatttttt aaatacccgc gagaaataga aaaaccaaca gttgatcgtc ttgcgaccga 3960 cggtggcgat aggcatccgg gtggtgctca aaagcagctt cgcctggctg atacgttggt 4020 cctcgcgcca gcttaagacg ctaatcccta actgctggcg gaaaagatgt gacagacgcg 4080 acggcgacaa gcaaacatgc tgtgcgacgc tggcgatatc aaaattgctg tctgccaggt 4140 gtactgacaa gatcgctgat gcctcgcgta cccgattatc catcggtgga tggagcgact 4200 cgttaatcgc ttccatgcgc cgcagtaaca attgctcaag cagatttatc gccagcagct 4260 ccgaatagcg cccttcccct tgcccggcgt taatgatttg cccaaacagg tcgctgaaat 4320 gcggctggtg cgcttcatcc gggcgaaaga accccgtatt ggcaaatatt gacggccagt 4380 taagccattc atgccagtag gcgcgcggac gaaagtaaac ccactggtga taccattcgc 4440 gagcctccgg atgacgaccg tagtgatgaa tctctcctgg cgggaacagc aaaatatcac 4500 ccggtcggca aacaaattct cgtccctgat ttttcaccac cccctgaccg cgaatggtga 4560 gattgagaat ataacctttc attcccagcg gtcggtcgat aaaaaaatcg agataaccgt 4620 cggcgttaaa tggcctcaat gatgggcatt cccgccacca cccggcagca aaacgagtat 4680 ggggatcatt ttgcgcttca gccatacttt tcatactccc gccattcaga 9 4731 <210> 3 <211> 4734 <212> DNA <213> Artifi ci al Sequence <220> <223> ppreTOP2-TFAM sequence <400> 3 aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct 60 tctcgctaac caaaccggta accccgctta ttaaaagcat agcgggacca tctgtaacaa 120 aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg 180 attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg 240 atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg tttttttggg 300 ctaacaggag gaattaacca tgggccacca ccatcaccac caccatcatc atcatggcgg 360 cagatctttt tccagcatgg gtagctatcc aaagaaacct atgagttcat accttcgatt 420 cagctaccca ttccacagaa aatttaaagc taaacaccca gatgcaaaac tttcagaatt 480 attgcagccc ggttaggaaa tgtggaggga gctaccagaa gcagaaaaaa aggtttatga 540 agctgatttt aaagctgagt ggaaagcata caaagaagct gtgagcaagt ataaagagca 600 agtcagctga gctaactcca tgggtatgga gaaggaggcc cggcagagac ggttaaaaaa 660 gaaagcactg gtaaagagaa gagaattaat tttgcttgga aaaccaaaaa gacctcgttc 720 agcatataac atttatgtat ctgaaagctt ccaggaggca aaggatgatt cggctcaggg 780 aaaattgaag cttgtaaatg aggcttggaa aaatctgtct cctgaggaaa agcaggcata 840 tattcagctt gc taaagatg ataggattcg ttacgacaat gaaatgaagt cttgggaaga 900 gcagatggct gaagttggac gaagtgatct catccgtcga agtgtgaaac gatccggaga 960 catctctgag cattaaggaa gacggagttg tcattggaat tcgaagcttg ggcccgaaca 1020 aaaactcatc tcagaagagg atctgaatag cgccgtcgac catcatcatc atcatcattg 1080 agtttaaacg gtctccagct tggctgtttt ggcggatgag agaagatttt cagcctgata 1140 cagattaaat cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc 1200 gcggtggtcc cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt 1260 agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc 1320 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag 1380 taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg 1440 ccgccataaa ggcaggacgc tcaaattaag ctgccaggca tcctgacgga cagaaggcca 1500 tggccttttt gcgtttctac aaactctttt tgtttatttt tctaaataca ttcaaatatg 1560 tgagacaata tatccgctca accctgataa atgcttcaat aatattgaaa aaggaagagt 1620 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 1680 gtttttgctc acccagaaac gctggtgaaa ctgaagatca gtaaaagatg gttgggtgca 1740 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 1800 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 1860 cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 1920 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 1980 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 2040 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 2100 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 2160 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 2220 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 2280 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 2340 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 2400 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 2460 agcattggta tcactgatta actgtcagac caagtttact catatatact ttagattgat 2520 ttaaaacttc atttttaatt taaaa ggatc taggtgaaga tcctttttga taatctcatg 2580 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 2640 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 2700 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 2760 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta 2820 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 2880 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 2940 aggcgcagcg ttaccggata gtcgggctga acggggggtt cgtgcacaca gcccagcttg 3000 cctacaccga gagcgaacga actgagatac ctacagcgtg agctatgaga aagcgccacg 3060 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 3120 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 3180 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 3240 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 3300 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 3360 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 3420 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg 3480 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 3540 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 3600 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 3660 gaggttttca gcatgtgtca ccgtcatcac cgaaacgcgc gaggcagcag atcaattcgc 3720 gcgcgaaggc gaagcggcat gcataatgtg cctgtcaaat ggacgaagca gggattctgc 3780 aaaccctatg ctactccgtc aagccgtcaa ttgtctgatt cgttaccaat tatgacaact 3840 tgacggctac atcattcact ttttcttcac aaccggcacg gaactcgctc gggctggccc 3900 cggtgcattt tttaaatacc cgcgagaaat agagttgatc gtcaaaacca acattgcgac 3960 cgacggtggc gataggcatc cgggtggtgc tcaaaagcag cttcgcctgg ctgatacgtt 4020 ggtcctcgcg ccagcttaag acgctaatcc ctaactgctg gcggaaaaga tgtgacagac 4080 caagcaaaca gcgacggcga tgctgtgcga cgctggcgat atcaaaattg ctgtctgcca 4140 ggtgatcgct gatgtactga caagcctcgc gtacccgatt atccatcggt ggatggagcg 4200 actcgttaat cgcttccatg cgccgcagta acaatt GCTC aagcagattt atcgccagca 4260 gctccgaata gcgcccttcc ccttgcccgg cgttaatgat ttgcccaaac aggtcgctga 4320 gtgcgcttca aatgcggctg tccgggcgaa agaaccccgt attggcaaat attgacggcc 4380 agttaagcca ttcatgccag taggcgcgcg gacgaaagta aacccactgg tgataccatt 4440 cggatgacga cgcgagcctc ccgtagtgat gaatctctcc tggcgggaac agcaaaatat 4500 cacccggtcg gcaaacaaat tctcgtccct gatttttcac caccccctga ccgcgaatgg 4560 tgagattgag aatataacct ttcattccca gcggtcggtc gataaaaaaa tcgagataac 4620 cgttggcctc aatcggcgtt aaacccgcca ccagatgggc attaaacgag tatcccggca 4680 gcaggggatc attttgcgct tcagccatac ttttcatact cccgccattc agag 4734 <210> 4 <211> 60 <212> DNA <213> Artifi ci al Sequence <220> <223> Direct primer for cloning the pre-Scission target in pPRETOP2-Tfam containing the targets of Bgl II and NheI <400> 4 ttttagatct ctggaagttc tgttccaggg gcccgctagc ttttccagca tgggtagcta 60 <210> 5 <211> 27 <212> DNA <213> Artifi ci al Sequence <220> <223> Reverse primer for cloning the pre-Scission target in pPRETOP2-Tfam <400> 5 catacccatc agctgacttg gagttag 27 <210> 6 <211> 4764 <212> DNA <213> Artifi ci al Sequence <220> <223> Sequence of plasmid ppreTOP3-Tfam <400> 6 aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct 60 tctcgctaac caaaccggta accccgctta ttaaaagcat agcgggacca tctgtaacaa 120 aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg 180 attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg 240 atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg tttttttggg 300 ctaacaggag gaattaacca tgggccacca ccatcaccac caccatcatc atcatggcgg 360 cagatctctg gaagttctgt tccaggggcc cgctagcttt tccagcatgg gtagctatcc 420 aaagaaacct atgagttcat accttcgatt ttccacagaa cagctaccca aatttaaagc 480 taaacaccca gatgcaaaac tttcagaatt ggttaggaaa attgcagccc tgtggaggga 540 gcagaaaaaa gctaccagaa aggtttatga agctgatttt aaagctgagt ggaaagcata 600 gtgagcaagt caaagaagct ataaagagca gctaactcca tgggtatgga agtcagctga 660 gaaggaggcc cggcagagac ggttaaaaaa gaaagcactg gtaaagagaa gagaattaat 720 aaaccaaaaa tttgcttgga gacctcgttc agcatataac atttatgtat ctgaaagctt 780 ccaggaggca aaggatgatt cggctcaggg aaaattgaag cttgtaaatg aggcttggaa 840 aaatctgtct cc tgaggaaa agcaggcata tattcagctt gctaaagatg ataggattcg 900 ttacgacaat gaaatgaagt cttgggaaga gcagatggct gaagttggac gaagtgatct 960 agtgtgaaac catccgtcga gatccggaga catctctgag cattaaggaa gacggagttg 1020 tcattggaat tcgaagcttg ggcccgaaca aaaactcatc tcagaagagg atctgaatag 1080 cgccgtcgac catcatcatc atcatcattg agtttaaacg gtctccagct tggctgtttt 1140 ggcggatgag agaagatttt cagcctgata cagattaaat cagaacgcag aagcggtctg 1200 ataaaacaga atttgcctgg cggcagtagc gcggtggtcc cacctgaccc catgccgaac 1260 tcagaagtga aacgccgtag cgccgatggt agtgtggggt ctccccatgc gagagtaggg 1320 aactgccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat 1380 ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa 1440 cgttgcgaag caacggcccg gagggtggcg ggcaggacgc ctgccaggca ccgccataaa 1500 cagaaggcca tcaaattaag tcctgacgga tggccttttt gcgtttctac aaactctttt 1560 tctaaataca tgtttatttt ttcaaatatg tatccgctca tgagacaata accctgataa 1620 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 1680 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 1740 ctgaagatca gtaaaagatg gttgggtgca cgagtgggtt acatcgaact ggatctcaac 1800 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 1860 aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt 1920 actattctca cgccgcatac gaatgacttg gttgagtact caccagtcac agaaaagcat 1980 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 2040 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 2100 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 2160 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa 2220 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag 2280 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 2340 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 2400 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 2460 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 2520 catatatact caagtttact ttaga ttgat ttaaaacttc atttttaatt taaaaggatc 2580 tcctttttga taggtgaaga taatctcatg accaaaatcc cttaacgtga gttttcgttc 2640 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 2700 gctgcttgca cgcgtaatct aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 2760 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 2820 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 2880 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 2940 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 3000 cgtgcacaca acggggggtt gcccagcttg gagcgaacga cctacaccga actgagatac 3060 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 3120 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 3180 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 3240 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 3300 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg 3360 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag 3420 cagtgagcga cgcagcgagt ggaagcggaa gagcgcctga tgcggtattt tctccttacg 3480 catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc 3540 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc 3600 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 3660 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 3720 cgaaacgcgc gaggcagcag atcaattcgc gcgcgaaggc gaagcggcat gcataatgtg 3780 cctgtcaaat ggacgaagca gggattctgc aaaccctatg ctactccgtc aagccgtcaa 3840 ttgtctgatt cgttaccaat tatgacaact tgacggctac atcattcact ttttcttcac 3900 aaccggcacg gaactcgctc gggctggccc cggtgcattt tttaaatacc cgcgagaaat 3960 agagttgatc gtcaaaacca acattgcgac cgacggtggc gataggcatc cgggtggtgc 4020 tcaaaagcag cttcgcctgg ctgatacgtt ggtcctcgcg ccagcttaag acgctaatcc 4080 gcggaaaaga ctaactgctg gcgacggcga tgtgacagac tgctgtgcga caagcaaaca 4140 cgctggcgat atcaaaattg ctgtctgcca ggtgatcgct gatgtactga caagcctcgc 4200 gtacccgatt atccatcggt ggatggagcg actcgt taat cgcttccatg cgccgcagta 4260 aagcagattt acaattgctc atcgccagca gctccgaata gcgcccttcc ccttgcccgg 4320 cgttaatgat ttgcccaaac aggtcgctga aatgcggctg tccgggcgaa gtgcgcttca 4380 agaaccccgt attggcaaat attgacggcc agttaagcca ttcatgccag taggcgcgcg 4440 gacgaaagta aacccactgg tgataccatt cgcgagcctc cggatgacga ccgtagtgat 4500 gaatctctcc tggcgggaac agcaaaatat cacccggtcg gcaaacaaat tctcgtccct 4560 caccccctga gatttttcac ccgcgaatgg tgagattgag aatataacct ttcattccca 4620 gcggtcggtc gataaaaaaa tcgagataac cgttggcctc aatcggcgtt aaacccgcca 4680 ccagatgggc attaaacgag tatcccggca gcaggggatc attttgcgct tcagccatac 4740 ttttcatact cccgccattc agag 4764 <210> 7 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Direct primer for cloning the sequence coding for GST in pPRETOP3-Tfam <400> 7 catcatcatg gcggcagatc ttcccctata ctagg 35 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for cloning the sequence coding for GST in pPRETOP3-Tfam <400> 8 aaagctagcg ggcccctgga acagaacttc 30 <210> 9 <211> 5421 <212> DNA <213> Artificial Sequence <220> <223> Plasmid sequence ppreTOP4-Tfam <400> 9 aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct 60 tctcgctaac caaaccggta accccgctta ttaaaagcat agcgggacca tctgtaacaa 120 aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg 180 attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg 240 atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg tttttttggg 300 ctaacaggag gaattaacca tgggccacca ccatcaccac caccatcatc atcatggcgg 360 cagatcttcc cctatactag gttattggaa aattaagggc cttgtgcaac ccactcgact 420 tcttttggaa tatcttgaag aaaaatatga agagcatttg tatgagcgcg atgaaggtga 480 taaatggcga aacaaaaagt ttgaattggg tttggagttt cccaatcttc cttattatat 540 tgatggtgat gttaaattaa ggccatcata cacagtctat cgttatatag ctgacaagca 600 caacatgttg ggtggttgtc caaaagagcg tgcagagatt tcaatgcttg aaggagcggt 660 tttggatatt agatacggtg tttcgagaat tgcatatagt aaagactttg aaactctcaa 720 agttgatttt cttagcaagc tacctgaaat gctgaaaatg ttcgaagatc gtttatgtca 780 taaaacatat ttaaatggtg atcatgtaac ccatcctgac ttcatgttgt atgacgctct 840 tgatgttgtt tt atacatgg acccaatgtg cctggatgcg ttcccaaaat tagtttgttt 900 taaaaaacgt attgaagcta tcccacaaat tgataagtac ttgaaatcca gcaagtatat 960 agcatggcct ttgcagggct ggcaagccac gtttggtggt ggcgaccatc ctccaaaatc 1020 ggatctggaa gttctgttcc aggggcccgc tagcttttcc agcatgggta gctatccaaa 1080 gaaacctatg agttcatacc ttcgattttc cacagaacag ctacccaaat ttaaagctaa 1140 acacccagat gcaaaacttt cagaattggt taggaaaatt gcagccctgt ggagggagct 1200 gaaaaaaagg accagaagca tttatgaagc tgattttaaa gctgagtgga aagcatacaa 1260 agaagctgtg agcaagtata aagagcagct aactccaagt cagctgatgg gtatggagaa 1320 ggaggcccgg cagagacggt taaaaaagaa agcactggta aagagaagag aattaatttt 1380 gcttggaaaa ccaaaaagac ctcgttcagc atataacatt tatgtatctg aaagcttcca 1440 ggaggcaaag gatgattcgg ctcagggaaa attgaagctt gtaaatgagg cttggaaaaa 1500 tctgtctcct gaggaaaagc aggcatatat tcagcttgct aaagatgata ggattcgtta 1560 atgaagtctt cgacaatgaa gggaagagca gatggctgaa gttggacgaa gtgatctcat 1620 ccgtcgaagt gtgaaacgat ccggagacat ctctgagcat taaggaagac ggagttgtca 1680 ttggaattcg aagcttgggc ccgaacaaaa actcatctca gaagaggatc tgaatagcgc 1740 cgtcgaccat catcatcatc atcattgagt ttaaacggtc tccagcttgg ctgttttggc 1800 ggatgagaga agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata 1860 aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca 1920 gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac 1980 tgccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg 2040 ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg atttgaacgt 2100 tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg ccataaactg ccaggcatca 2160 aattaagcag aaggccatcc tgacggatgg cctttttgcg tttctacaaa ctctttttgt 2220 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg 2280 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt 2340 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta 2400 aaagatgctg aagatcagtt gtgggttaca gggtgcacga tcgaactgga tctcaacagc 2460 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa 2520 gttctgctat gtggcgcggt attat cccgt gttgacgccg ggcaagagca actcggtcgc 2580 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt 2640 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact 2700 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac 2760 aacatggggg atcatgtaac tcgccttgat cgttgggaac tgaagccata cggagctgaa 2820 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta 2880 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg 2940 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat 3000 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt 3060 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga 3120 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa 3180 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag 3240 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac 3300 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc 3360 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat 3420 caactctttt caagagctac actggcttca tccgaaggta gcagagcgca gataccaaat 3480 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct 3540 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt 3600 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg 3660 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta 3720 cagcgtgagc tatgagaaag cgccacgctt gaaaggcgga cccgaaggga caggtatccg 3780 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg 3840 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc 3900 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg 3960 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat 4020 ccgcctttga aaccgtatta gtgagctgat accgctcgcc gcagccgaac gaccgagcgc 4080 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat 4140 ctgtgcggta tttcacaccg catatggtgc actctcagta tgatgccgca caatctgctc 4200 tagttaagcc agtatacact ccgctatcgc tacgtg actg ggtcatggct gcgccccgac 4260 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca 4320 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 4380 aacgcgcgag gcagcagatc aattcgcgcg cgaaggcgaa gcggcatgca taatgtgcct 4440 gtcaaatgga cgaagcaggg attctgcaaa ccctatgcta ctccgtcaag ccgtcaattg 4500 tctgattcgt taccaattat gacaacttga cggctacatc attcactttt tcttcacaac 4560 cggcacggaa ctcgctcggg ctggccccgg tgcatttttt aaatacccgc gagaaataga 4620 aaaaccaaca gttgatcgtc ttgcgaccga cggtggcgat aggcatccgg gtggtgctca 4680 aaagcagctt cgcctggctg atacgttggt cctcgcgcca gcttaagacg ctaatcccta 4740 actgctggcg gaaaagatgt gacagacgcg acggcgacaa gcaaacatgc tgtgcgacgc 4800 tggcgatatc aaaattgctg tctgccaggt gatcgctgat gtactgacaa gcctcgcgta 4860 cccgattatc catcggtgga tggagcgact cgttaatcgc ttccatgcgc cgcagtaaca 4920 attgctcaag cagatttatc gccagcagct ccgaatagcg cccttcccct tgcccggcgt 4980 taatgatttg cccaaacagg tcgctgaaat gcggctggtg cgcttcatcc gggcgaaaga 5040 accccgtatt ggcaaatatt gacggccagt taagccattc to tgccagtag gcgcgcggac 5100 gaaagtaaac ccactggtga taccattcgc gagcctccgg atgacgaccg tagtgatgaa 5160 tctctcctgg cgggaacagc aaaatatcac ccggtcggca aacaaattct cgtccctgat 5220 ttttcaccac cccctgaccg cgaatggtga gattgagaat ataacctttc attcccagcg 5280 gtcggtcgat aaaaaaatcg agataaccgt tggcctcaat cccgccacca cggcgttaaa 5340 gatgggcatt aaacgagtat cccggcagca ggggatcatt ttgcgcttca gccatacttt tcatactccc 9 5421 5400 gccattcaga <210> 10 <211> 90 <212> DNA <213> Artifi ci al Sequence <220> <223> Direct primer with the sequence coding for 10 histidines and for the enterokinase cut-off target <400> 10 gaagttctgt tccaggggcc ccatcaccat caccatcacc atcaccatca cgatgacgat 60 gacaagtttt ccagcatggg tagctatcca 90 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer that hybridizes with the sequence coding for the pre-Scission cutoff target <400> 11 aaagctagcg ggcccctgga acagaacttc 30 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Direct primer to distinguish pPRETOP4-Tfam from pTOP-Tfam <400> 12 ctaggctgga agttctgttc caggggcccg 30 <210> 13 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer to distinguish pPRETOP4-Tfam from pTOP-Tfam <400> 13 ttttgaattc ctctttatac ttgctcacag cttcttt 37 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Direct primer to check the pTOP-Tfam vector <400> 14 gaccaaagcc atgacaaaaa cg 22 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer to check the pTOP-Tfam vector <400> 15 tcgacggcgc tattcagatc 20 <210> 16 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Direct cloning primer of PCBP1 in pTOP <400> 16 cacgatgacg atgacaagga tgccggtgtg actgaa 36 <210> 17 <211> 33 <212> DNA <213> Artifi ci al Sequence <220> <223> PCBP1 reverse cloning primer in pTOP <400> 17 atgacaactc cgtcttccct agctgcaccc cat 33 <210> 18 <211> 5918 <212> DNA <213> Artifi ci al Sequence <220> <223> Sequence of pTOP-PCBP1 <400> 18 aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct 60 tctcgctaac caaaccggta accccgctta ttaaaagcat agcgggacca tctgtaacaa 120 aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg 180 attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg 240 atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg ttttttgggc 300 taacaggagg aattaaccat gggccaccac catcaccacc accatcatca tcatggcggc 360 agatcttccc ctatactagg ttattggaaa attaagggcc ttgtgcaacc cactcgactt 420 atcttgaaga cttttggaat aaaatatgaa gagcatttgt atgagcgcga tgaaggtgat 480 aaatggcgaa acaaaaagtt tgaattgggt ttggagtttc ccaatcttcc ttattatatt 540 gatggtgatg ttaaattaac acagtctatg gccatcatac gttatatagc tgacaagcac 600 aacatgttgg gtggttgtcc aaaagagcgt gcagagattt caatgcttga aggagcggtt 660 gatacggtgt ttggatatta gcatatagta ttcgagaatt aagactttga aactctcaaa 720 gttgattttc ttagcaagct acctgaaatg ctgaaaatgt tcgaagatcg tttatgtcat 780 aaaacatatt taaatggtga tcatgtaacc catcctgact tcatgttgta tgacgctctt 840 gatgttgttt t atacatgga cccaatgtgc ctggatgcgt tcccaaaatt agtttgtttt 900 aaaaaacgta ttgaagctat cccacaaatt gataagtact tgaaatccag caagtatata 960 gcatggcctt tgcagggctg gcaagccacg tttggtggtg gcgaccatcc tccaaaatcg 1020 ttctgttcca gatctggaag ggggccccat caccatcacc atcaccatca ccatcacgat 1080 gacgatgaca aggatgccgg tgtgactgaa agtggactaa atgtgactct caccattcgg 1140 acggaaagga cttcttatgc atcattggga agtaggaagc agaaagggga gtcggttaag 1200 aggatccgcg aggagagtgg cgcgcggatc aacatctcgg aggggaattg tccggagaga 1260 atcatcactc tgaccggccc caccaatgcc atctttaagg ctttcgctat gatcatcgac 1320 aagctggagg aagatatcaa cagctccatg accaacagta ccgcggccag caggcccccg 1380 gtcaccctga ggctggtggt gccggccacc cagtgcggct ccctgattgg gaaaggcggg 1440 tgtaagatca aagagatccg cgagagtacg ggggcgcagg tccaggtggc gggggatatg 1500 ctgcccaact ccaccgagcg ggccatcacc atcgctggcg tgccgcagtc tgtcaccgag 1560 tgtgtcaagc agatttgcct ggtcatgctg gagacgctct cccagtctcc gcaagggaga 1620 ttccgtacca gtcatgacca gcccatgccg gccagctccc cagtcatctg cgcgggcggc 1680 caagatcggt gcagcgacg c tgcgggctac ccccatgcca cccatgacct ggagggacca 1740 cctctagatg cctactcgat tcaaggacaa cacaccattt ctccgctcga tctggccaag 1800 tggcaagaca ctgaaccagg acagtctcac tttgccatga tgcacggcgg gaccggattc 1860 gccggaattg actccagctc tccagaggtg aaaggctatt gggcaagttt ggatgcatct 1920 cccatgaact actcaaacca caccattcca aataacttaa ttggctgcat aatcgggcgc 1980 acattaatga caaggcgcca gatccgccag atgtccgggg cccagatcaa aattgccaac 2040 ccagtggaag gctcctctgg taggcaggtt actatcactg gctctgctgc cagtattagt 2100 atctaatcaa ctggcccagt tgccaggctt tcctctgaga agggcatggg gtgcagctag 2160 ggaagacgga gttgtcattg gaattcgaag cttgggcccg aacaaaaact catctcagaa 2220 gaggatctga atagcgccgt cgaccatcat catcatcatc attgagttta aacggtctcc 2280 ttttggcgga agcttggctg tgagagaaga ttttcagcct gatacagatt aaatcagaac 2340 gcagaagcgg tctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg 2400 accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc 2460 atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg 2520 gcctttcgtt ttatctgttg TTTG tcggtg aacgctctcc tgagtaggac aaatccgccg 2580 ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca 2640 taaactgcca ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt 2700 tttttgttta ctacaaactc tttttctaaa tacattcaaa tatgtatccg ctcatgagac 2760 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 2820 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 2880 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 2940 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 3000 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc 3060 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 3120 tcacagaaaa gcatcttacg gatggcatga attatgcagt cagtaagaga gctgccataa 3180 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 3240 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 3300 agccatacca agctgaatga aacgacgagc gtgacaccac gcaatggcaa gatgcctgta 3360 caaactatta caacgttgcg actggcgaac tacttactct agcttcccgg caacaattaa 3420 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 3480 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 3540 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 3600 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 3660 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 3720 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 3780 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 3840 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 3900 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 3960 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 4020 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 4080 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 4140 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag acgacctaca cttggagcga 4200 ccgaactgag atacctacag cgtgagctat Gagaa agcgc cacgcttccc gaagggagaa 4260 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 4320 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 4380 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 4440 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 4500 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 4560 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt 4620 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa 4680 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt 4740 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct 4800 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt 4860 ttcaccgtca tcaccgaaac gcgcgaggca gcagatcaat tcgcgcgcga aggcgaagcg 4920 gcatgcataa tgtgcctgtc aaatggacga agcagggatt ctgcaaaccc tatgctactc 4980 cgtcaagccg tcaattgtct gattcgttac caattatgac aacttgacgg ctacatcatt 5040 cactttttct tcacaaccgg cacggaactc gctcgggctg gccccggtgc attttttaaa 5100 tacccgcgag aaatagagtt gatcgtcaaa accaacattg cgaccgacgg tggcgatagg 5160 catccgggtg gtgctcaaaa gcagcttcgc ctggctgata cgttggtcct cgcgccagct 5220 taagacgcta atccctaact gctggcggaa aagatgtgac agacgcgacg gcgacaagca 5280 aacatgctgt gcgacgctgg cgatatcaaa attgctgtct gccaggtgat cgctgatgta 5340 ctgacaagcc tcgcgtaccc gattatccat cggtggatgg agcgactcgt taatcgcttc 5400 catgcgccgc agtaacaatt gctcaagcag atttatcgcc agcagctccg aatagcgccc 5460 ttccccttgc ccggcgttaa tgatttgccc aaacaggtcg ctgaaatgcg gctggtgcgc 5520 ttcatccggg cgaaagaacc ccgtattggc aaatattgac ggccagttaa gccattcatg 5580 ccagtaggcg cgcggacgaa agtaaaccca ctggtgatac cattcgcgag cctccggatg 5640 acgaccgtag tgatgaatct ctcctggcgg gaacagcaaa atatcacccg gtcggcaaac 5700 aaattctcgt ccctgatttt tcaccacccc ctgaccgcga atggtgagat tgagaatata 5760 acctttcatt cccagcggtc ggtcgataaa aaaatcgaga taaccgttgg cctcaatcgg 5820 cgttaaaccc gccaccagat gggcattaaa cgagtatccc ggcagcaggg gatcattttg 5880 atacttttca cgcttcagcc tactcccgcc attcagag 5918 <210> 19 <211> 5460 <212> DNA <213> Artifi ci al Sequence <220> <223> Sequence of plasmid pTOP-Tfam <400> 19 aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct 60 tctcgctaac caaaccggta accccgctta ttaaaagcat agcgggacca tctgtaacaa 120 aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg 180 attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg 240 atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg tttttttggg 300 ctaacaggag gaattaacca tgggccacca ccatcaccac caccatcatc atcatggcgg 360 cagatcttcc cctatactag gttattggaa aattaagggc cttgtgcaac ccactcgact 420 tcttttggaa tatcttgaag aaaaatatga agagcatttg tatgagcgcg atgaaggtga 480 taaatggcga aacaaaaagt ttgaattggg tttggagttt cccaatcttc cttattatat 540 tgatggtgat gttaaattaa ggccatcata cacagtctat cgttatatag ctgacaagca 600 caacatgttg ggtggttgtc caaaagagcg tgcagagatt tcaatgcttg aaggagcggt 660 tttggatatt agatacggtg tttcgagaat tgcatatagt aaagactttg aaactctcaa 720 agttgatttt cttagcaagc tacctgaaat gctgaaaatg ttcgaagatc gtttatgtca 780 taaaacatat ttaaatggtg atcatgtaac ccatcctgac ttcatgttgt atgacgctct 840 tgatgttgtt t tatacatgg acccaatgtg cctggatgcg ttcccaaaat tagtttgttt 900 taaaaaacgt attgaagcta tcccacaaat tgataagtac ttgaaatcca gcaagtatat 960 agcatggcct ttgcagggct ggcaagccac gtttggtggt ggcgaccatc ctccaaaatc 1020 ggatctggaa gttctgttcc aggggcccca tcaccatcac catcaccatc accatcacga 1080 tgacgatgac aagttttcca gcatgggtag ctatccaaag aaacctatga gttcatacct 1140 tcgattttcc acagaacagc tacccaaatt taaagctaaa cacccagatg caaaactttc 1200 agaattggtt aggaaaattg cagccctgtg gagggagcta ccagaagcag aaaaaaaggt 1260 ttatgaagct gattttaaag ctgagtggaa gaagctgtga agcatacaaa gcaagtataa 1320 agagcagcta actccaagtc agctgatggg tatggagaag gaggcccggc agagacggtt 1380 gcactggtaa aaaaaagaaa agagaagaga attaattttg cttggaaaac caaaaagacc 1440 tcgttcagca tataacattt atgtatctga aagcttccag gaggcaaagg atgattcggc 1500 tcagggaaaa ttgaagcttg taaatgaggc ttggaaaaat ctgtctcctg aggaaaagca 1560 ggcatatatt cagcttgcta aagatgatag gattcgttac gacaatgaaa tgaagtcttg 1620 ggaagagcag atggctgaag ttggacgaag tgatctcatc cgtcgaagtg tgaaacgatc 1680 cggagacatc tctgagcat t aaggaagacg gagttgtcat tggaattcga agcttgggcc 1740 cgaacaaaaa ctcatctcag aagaggatct gaatagcgcc gtcgaccatc atcatcatca 1800 tcattgagtt taaacggtct ccagcttggc tgttttggcg gatgagagaa gattttcagc 1860 ttaaatcaga ctgatacaga acgcagaagc ggtctgataa aacagaattt gcctggcggc 1920 agtagcgcgg tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc 1980 gatggtagtg tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg 2040 aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct 2100 cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg 2160 gtggcgggca ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct 2220 gacggatggc ctttttgcgt ttctacaaac tctttttgtt tatttttcta aatacattca 2280 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 2340 gtattcaaca aagagtatga tttccgtgtc gcccttattc ccttttttgc ggcattttgc 2400 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 2460 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 2520 cgccccgaag aacgttttcc AATG atgagc acttttaaag ttctgctatg tggcgcggta 2580 ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 2640 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 2700 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 2760 gaccgaagga acgatcggag gctaaccgct tttttgcaca acatggggga tcatgtaact 2820 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 2880 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 2940 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 3000 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 3060 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 3120 cggggagtca atctacacga ggcaactatg gatgaacgaa atagacagat cgctgagata 3180 tgattaagca ggtgcctcac ttggtaactg tcagaccaag tttactcata tatactttag 3240 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 3300 aaatccctta ctcatgacca acgtgagttt tcgttccact ccccgtagaa gagcgtcaga 3360 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 3420 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 3480 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg 3540 accacttcaa tagttaggcc gaactctgta gcaccgccta catacctcgc tctgctaatc 3600 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 3660 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 3720 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 3780 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 3840 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 3900 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 3960 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 4020 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag 4080 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa 4140 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 4200 atatggtgca ctctcagtac aatctgctct gatgc cgcat agttaagcca gtatacactc 4260 cgctatcgct acgtgactgg gtcatggctg cccgccaaca cgccccgaca cccgctgacg 4320 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg 4380 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagcagatca 4440 attcgcgcgc gaaggcgaag cggcatgcat aatgtgcctg tcaaatggac gaagcaggga 4500 ttctgcaaac cctatgctac tccgtcaagc cgtcaattgt ctgattcgtt accaattatg 4560 ggctacatca acaacttgac ttcacttttt cttcacaacc ggcacggaac tcgctcgggc 4620 tggccccggt gcatttttta aatacccgcg agaaatagag ttgatcgtca aaaccaacat 4680 tgcgaccgac ggtggcgata ggcatccggg tggtgctcaa aagcagcttc gcctggctga 4740 tacgttggtc ctcgcgccag cttaagacgc taatccctaa ctgctggcgg aaaagatgtg 4800 acagacgcga cggcgacaag caaacatgct gtgcgacgct ggcgatatca aaattgctgt 4860 ctgccaggtg atcgctgatg tactgacaag cctcgcgtac ccgattatcc atcggtggat 4920 ggagcgactc gttaatcgct tccatgcgcc gcagtaacaa ttgctcaagc agatttatcg 4980 ccagcagctc cgaatagcgc ccttcccctt gcccggcgtt aatgatttgc ccaaacaggt 5040 cgctgaaatg cggctggtgc gcttcatccg ggcgaaagaa ccccgtattg gcaaatattg 5100 aagccattca acggccagtt tgccagtagg cgcgcggacg aaagtaaacc cactggtgat 5160 accattcgcg agcctccgga tgacgaccgt agtgatgaat ctctcctggc gggaacagca 5220 aaatatcacc cggtcggcaa acaaattctc gtccctgatt tttcaccacc ccctgaccgc 5280 attgagaata gaatggtgag ttcccagcgg taacctttca tcggtcgata aaaaaatcga 5340 gataaccgtt ggcctcaatc ggcgttaaac ccgccaccag atgggcatta aacgagtatc 5400 ccggcagcag gggatcattt tgcgcttcag ccatactttt catactcccg ccattcagag 5460 <210> 20 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Direct primer of reamplification of the insert to be cloned in pTOP by CipCR <400> 20 tcaccatcac catcaccatc acgatgacga tgacaag 37 <210> 21 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of reamplification of the insert to be cloned in pTOP by CipCR <400> 21 gcccaagctt cgaattccaa tgacaactcc gtcttcctta 40 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Part 5 of the direct primer for cloning the insect by CipCR <400> 22 cacgatgacg atgacaag 18 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Part 5 of the reverse primer for cloning the insert by CipCR <400> 23 atgacaactc cgtcttcctt a 21