WO2012113959A1 - pTOP: NOVEL VECTOR FOR THE EXPRESSION AND PURIFICATION OF PROTEINS - Google Patents

Abstract

The invention relates to a novel gene expression vector (1) that can be used for marking, expressing and purifying proteins, to the protein expression and purification method (2) associated with the vector and to a kit (3) 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 protein screening, expression and purification, (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 to control 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-pD-1 - thiogalactopyranoside, a lactose analogue but more advantageous because their levels are maintained because it does not degrade during cell culture) in cell cultures transformed with pGEX, and the ara (arabinose) operon that is induced by adding arabinose in cell cultures transformed with pBAD. In addition, both systems allow the marking and obtaining of 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 simian virus 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 case of pBAD.

 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 a "Hgation independent cloning" method, which, unlike other methods used in cloning in pGEX and in pBAD, prevents the addition of extra residues from the cloning stage to the protein of interest. The elements presented by the fusion protein resulting from expression in the p-CAL-n-FLAG vector have different uses: the CBP allows affinity purification of the fusion protein in a calmodulin resin. On the other hand, 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 It is convenient to release the protein thus obtained from the CBP marker by cutting with the Thrombin protease, since in this type of immunoprecipitation studies the presence of 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 obtaining 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 can perform more washes and greater astringency to selectively remove other unwanted cellular proteins that also have 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 an O 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, d marker. a nucleotide sequence encoding a protease recognition amino acid sequence P,

 and. a nucleotide sequence encoding an O 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) a 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; (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 markers M, N and O 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 marker peptide or hemagglutinin 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 protease Pre- Scission, and the amino acid sequence Q is the recognition target of the Enterokinase protease.

 The Tev protease is a protease of the Tobacco Etch Virus. The HRV 3C protease is a 3C protease of the human rhinovirus type 14. The ULP1 protease ("Ubl-specific protease 1") is a protease of the Ubis family ("ubiquitin-like proteins"), of the yeast Saccharomyces cerevisiae ( S. Cerevisiae), which is involved in the rupture of SUMO ("small ubiquitin-like modifief ') 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. He Origin of replication can be from prokaryotes or eukaryotes. Preferably, it comprises the origin of replication of plasmid pUC.

 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 encoding a fluorescent protein, a gene encoding a luminescent protein, a gene encoding beta-galactosidase 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 SEQ 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 SEQ ID NO: 1 and which, although it may have a slightly different sequence, retains the functional characteristics of SEQ ID NO: 1, that is, the differences with the sequence of SEQ 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 ** \ ^m (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 cloning method by CiPCR, which in addition to allowing to obtain the different variants of The fusion protein (Figure 3), also allows obtaining the pure protein without extra amino acids, and also has other advantages such as: 1) simplify the cloning process, 2) not require high knowledge of genetic engineering, 3) avoid 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 ^' (SEQ 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 (SEQ ID NO: 22) + sequence-primer-direct-gene-interest 3 ^' . The reverse oligonucleotide starts at the 5 ^'end with the ^5' -atgacaactccgtcttcc-3 ^'(SEQ ID NO: 23) and then the sequence reverse primer designed to amplify the gene of interest without including the stop codon is added, since this is included in the part corresponding to the pTOP vector sequence, obtaining the complete reverse oligonucleotide: 5 ^' atgacaactccgtcttcctta (SEQ ID NO: 23) + sequence-primer-reverse-gene-interest 3 ^' .

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

 In oligonucleotide primers it may be of interest to add mutations

M optional in two regions:

- between the sequence that hybridizes with the vector and that of priming for the amplification of the gene of interest to be inserted into pTOP - within the primer sequences designed for the 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 hybrid PCR 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 coincides with that of a fragment of the pTOP vector from the nucleotide at position 1.093 of SEQ 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 (SEQ ID NO: 20): 5 ^' tcaccatcaccatcaccatcacgatgacgatgacaag3 ^' , coinciding with the sequence of the pTOP vector (SEQ 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 SEQ ID NO: 1. An example of the 40 nucleotide reverse primer could be (SEQ ID NO: 21): 5 ^' gcccaagcttcgaattccaatgacaactccgtcttcctta3 ^' , whose sequence is complementary to that of the pTOP chain indicated in SEQ 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 position 1.133 of SEQ ID NO: 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 (SEQ ID NO : 22 and SEQ 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 melting temperatures, dimer formation, and other parameters of oligonucleotides that include hybridization sequences with the insert and with the vector and M mutations where appropriate, will help in the choice of the strategy to follow.

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 carrying the insert or 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, the step

(i) comprises at least two sub-stages:

i '. Enter the nucleotide sequence, gene construct or vector obtained in step (h) into a prokaryotic cell or into a eukaryotic cell,

i ". Cultivate the cell of stage (i ') in the presence of the substances necessary for the protein encoded to express the gene of interest cloned in the 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, Saccharomycetes Class, Order Saccharomycetales, Family Saccharomycetaceae and Genus Saccharomyces.

 Yeast P. Pastoris 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: 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 (j) 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 stage protein (j") from its binding to the stage ligand (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 (I I) (Ni) and / or cobalt (I I). 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 "Nsar", 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 immobilized nickel (II) and / or cobalt (II) 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 nucleotide sequence SEQ ID NO: 1 or a bioequivalent variant. More preferably, the pTOP vector is the nucleotide sequence SEQ ID NO: 1.

 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 oligonucleotide primers of direct and reverse reamplification of between 30 and 100 nucleotides, where the sequence of the direct primer coincides with the fragment of SEQ ID NO: 1 whose end 3 ^' is the nucleotide at position 1.093 of SEQ ID NO: 1 or the nucleotide at a position of up to 10 nucleotides above, and where the reverse primer sequence is complementary to SEQ ID NO: 1 so that its end 3 ^' is the nucleotide complementary to the nucleotide at position 1.094 of SEQ ID NO: 1 or the nucleotide at a position of up to 10 nucleotides later.

 The direct and reverse primers could be SEQ ID NO: 20 and SEQ ID NO:

21, respectively.

 Throughout the description and 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 features 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 (c); 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; TAA: 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. Western blot showing the development of arabinose induction of PCBP1 expression in the E. coli BL21 culture containing the pTOP-PCBP1 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 the plasmid pPreTOP1 -Tfam (SEQ ID NO: 2), where the Tfam gene cloned in pPreTOPI goes from nucleotide 365 to nucleotide 973 of SEQ ID NO: 2.

 Stage 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 II to obtain plasmid pPreTOP2-Tfam (SEQ ID NO: 3 ).

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

 The PTC-200 thermocycler program (MJ Research) consisted of 1 cycle at 95 ° C for 30 seconds, followed by 12 cycles consisting of three stages, at 95 ° C for 30 seconds, at 55 ° C for 1 minute, and 68 ° C for 5 minutes. After cooling on ice for 2 minutes, it was treated with 2 U of Dpnl for 2 hours at 37 ° C. Next, 2 μΙ of the reaction mixture was used to transform 50 μΙ of competent E. coli TOP10 cells (provided with the kit) by thermal shock. After 1 hour in orbital shaking at 200 rpm and 37 ° C 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 μΙ of sterile double-distilled water and visualized on a 1% agarose gel and staining with ethidium bromide (0.5 μg ml) . Subsequently, two reactions of 10 μΙ of total volume were prepared with each of the plasmids, respectively, for cutting with the restriction enzyme Bgl I I (Roche), according to the supplier's instructions. It was maintained at 37 ° C in a thermoblock for 1 hour and after deactivation of the enzyme for 20 minutes at 65 ° C in a thermoblock, the products were analyzed on a 1% agarose gel in TAE 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 Dye 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 Biosystems 3730x1 DNA Analyzer ™. The electropherograms were visualized with the Chromas Lite program (Copyright © 2005 Technelysium Pty Ltd), which confirmed the obtaining of plasmid pPreTOP2-Tfam.

Step 2. Simultaneous introduction of the Pre-Scission protease recognition sequence and the enzyme recognition sequence of Nhel restriction on 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 Pvul I (New England Biolabs) and binding with the T4 DNA ligase (Promega).

 Obtaining the insert

Primers were first designed properly to carry out the amplification of the insert by PCR. The direct primer (SEQ ID NO: 4), 5 ^' -tttt- agatct-ctggaagttctgttccaggggccc-gctagc-ttttccagcatgggtagcta-3 ^' was endowed with an extra sequence at the 5 ^' end containing the target sequence for the restriction enzyme Bgl II, between positions 5-10 of SEQ ID NO: 4, followed by the Pre-Scission protease target sequence, between positions 1 -34 of SEQ ID NO: 4, and the target sequence for Nhel restriction enzyme , between positions 35-40 of SEQ ID NO: 4, and at its 3 ^' end the sequence for hybridization with the DNA to be amplified was included. The reverse primer sequence (SEQ ID NO: 5), 5 ^' -catacccat-cagctg-acttggagttag-3 ^' hybrid in its entirety with the corresponding template sequence, which already contained the sequence for the restriction enzyme Pvul I, and which occupies positions 10-15 of SEQ ID NO: 5.

 For a total volume of 80 μΙ of reaction mixture, 16 μΙ of 5x PrimeSTAR buffer, direct and reverse primer 0.2 μΜ each, mixture of 0.2 mM dNTPs each, 200 ng of the pPreTOP2-Tfam mold were added cold (SEQ ID NO: 3), 0.8 μΙ of PrimeSTAR ™ HS DNA polymerase (Takara) and sterile double distilled water until the total volume is completed.

 The amplification program in a PTC-200 thermal cycler consisted of 1 cycle at 95 ° C for 5 minutes, followed by 30 cycles composed of three stages, at 95 ° C for 30 seconds, at 60 ° C for 30 seconds and at 72 ° C for 1 minute, and a final cycle at 72 ° C 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 Puré PCR Product Purification Kit (Roche), and elution with sterile double-distilled water, the amplified fragment was quantified by spectrophotometry in the Nanodrop 2000C. Two μg of the pure amplicon was cold mixed with 4 μΙ of 10x buffer, 0.4 μΙ of 100x BSA and 0.5 μΙ of each restriction enzyme Bgl II (NEB) and Pvul I (NEB) and double distilled water sterile up to 40 μΙ of total volume, and incubated at 37 ° C overnight in a thermoblock. The products were analyzed and purified again by the same procedure, quantified and stored at -20 ° C.

 Obtaining the linear vector

 Three μ9 of the pure circular pPreTOP2-Tfam plasmid (SEQ ID NO: 3) was cold mixed with 4 μΙ of 10x buffer, 0.4 μΙ of 100x BSA and 0.5 μΙ of each Bgl II restriction enzyme (NEB) and Pvul l (NEB) and sterile double-distilled water up to 40 μΙ of total volume and incubated at 37 ° C 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 (SEQ ID NO: 3), 2 μΙ of 10x buffer and 1 μΙ of T4 DNA ligase (NEB) and sterile double distilled water to complete 20 μΙ. The mixture was incubated at 16 ° C overnight in a thermal cycler in a cold chamber. Ninety μΙ of chemically competent E. coli DH5a bacteria were transformed with 10 μΙ of the ligation product and seeded on a plate of LB-ampicillin agar (100 μg 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 (SEQ ID NO: 3) was performed by direct sequencing of the plasmid of one of the positive clones, corroborating the obtaining of plasmid pPreTOP3-Tfam (SEQ ID NO: 6).

 Step 3. Introduction of the Glutathione S-transferase (GST) coding sequence in plasmid pPreTOP3-Tfam (SEQ ID NO: 6) to obtain plasmid pPreTOP4-Tfam (SEQ 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 μΙ containing 10 μΙ of PrimeSTAR 5X buffer, mixture of 0.2 mM dNTPs each, 0.2 μΜ of each of the oligonucleotide primers, 50 pg of plasmid pGEX-6P- 1 and 1, 25 U of PrimeSTAR ™ HS DNA 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-6p-1 mold region from which the insert was amplified (nucleotides 22-35 of SEQ ID NO : 7) and that of the region of the pPreTOP3-Tfam vector (SEQ ID NO: 6) where the insert was cloned (nucleotides 1-21 of SEQ ID NO: 7). The direct primer sequence was (SEQ ID NO: 7): 5 ^' - catcatcatggcggcagatct-tcccctatactagg-3 ^' and the reverse sequence (SEQ ID NO: 8) 5 ^' - aaagctagc-gggcccctggaacagaacttc -3 ^' , which included the coding sequence of the Pre-Scission protease target (nucleotides 1-9 of SEQ ID NO: 8). In all reactions, sterile double-distilled water was used as diluent. The amplification conditions in the PTC-200 thermal cycler consisted of 1 cycle of denaturation at 94 ° C for 30 seconds; 40 cycles composed of three stages, at 98 ° C for 10 seconds, at 54 ° C for 30 seconds, and 72 ° C for 45 seconds, and 1 final cycle at 72 ° C for 10 minutes. The PCR products were electrophoresed on a 1% agarose gel. The correct band was cut with a scalpel blade and the DNA fragments were extracted and purified with the High Puré 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 NanoDrop 2000C and stored at -20 ° C until use.

 CiPCR for the insertion of the GST coding sequence

 The CiPCR was performed on a PTC-200 thermal cycler in a final volume of

25 μΙ containing 5 μΙ of PrimeSTAR 5X buffer, 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 terminators of the elongation (at the 5 ^' end) by double hybridization of its two ends with the corresponding sequences of the vector in the place selected to clone the insert), 100 ng of plasmid pPreTOP3-Tfam (SEQ ID NO: 6) and 1.25 U of PrimeSTAR ™ HS DNA polymerase (Takara). The amplification conditions of the CiPCR were: 1 cycle of denaturation at 95 ° C for 5 minutes, 5 cycles composed of four stages, at 95 ° C for 1 minute, at 81 ° C for 30 seconds, at 65 ° C for 30 minutes seconds, and at 72 ° C for 6 minutes, 1 cycle of four stages, at 98 ° C for 30 seconds, at 81 ° C for 20 seconds, at 65 ° C for 20 seconds and at 72 ° C for 6 minutes. Next, another 10 cycles composed of four stages, at 98 ° C for 20 seconds, at 81 ° C for 15 seconds, at 65 ° C for 15 seconds and at 72 ° C for 6 minutes. Finally, 1 extension cycle at 72 ° C for 15 minutes. After ice cooling, the PCR product was treated with 2 U of the Dpnl enzyme (Fermentas) for 6 hours at 37 ° C, followed by heating denaturation, and after cooling renaturation the products were precipitated with sodium acetate / ethanol, air dried, diluted in 6 μΙ of sterile double-distilled water, which were used to thermally transform 60 μΙ of chemically competent cells E. coli TG-1. After seeding of the bacteria transformed in LB-agar plates containing ampicillin (100 g / 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 g / ml) and grown for 16 hours. The culture was cooled on ice, the cells were precipitated by centrifugation at 3,000 xg for 20 minutes, the supernatant was decanted and the plasmid was extracted from the GenElute (Sigma) plasmid purification kit, 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 g / 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 ° C. 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 pPreTOP4-Tfam (SEQ ID NO: 9).

 Step 4. Introduction of the 10 histidine epitope and the recognition sequence for the Enterokinase protease recognition in plasmid pPreTOP4-Tfam (SEQ ID NO: 9) to obtain plasmid pTOP-Tfam (SEQ ID NO: 19).

Insertion of epitope 10 histidines (10His or 10H) and the coding sequence of the cleavage site with enterokinase (EK) is held directly by CiPCR with primers: forward primer, (SEQ ID NO: 10) 5 ^'- gaagttctgttccaggggccc -catcaccatcaccatcaccatcaccatcac-gatgacgatgaca

ag-ttttccagcatgggtagctatcca-3 ', which contains the coding sequence of 10 His, between nucleotides 22-51, that of the EK target, between nucleotides 52-64, the sequence of 5 ^' ends, between nucleotides 1 - 21, is the termination signal of the polymerase elongation and that of the 3 ^' end is the hybridization sequence with the pPreTOP4-Tfam template (SEQ ID NO: 9); the reverse primer (SEQ ID NO: 1 1) with the sequence 5 ^' -aaagctagcgggcccctggaacagaacttc-3 ^' , hybrid in its entirety with the coding area of the Pre-Scission protease target of the template and insert. The final volume of the CiPCR mixture was 25 μΙ and contained 5 μΙ of 5X buffer, 0.2 mM dNTP mix each, 0.2 μΜ of each of the primers, 100 ng of plasmid pPreTOP4-Tfam and 1 , 25 U of PrimeSTAR ™ 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 ° C for 5 minutes, 5 amplification cycles composed of three stages, at 95 ° C for 1 minute, at 70 ° C for 30 seconds, at 72 ° C for 7 minutes. A cycle composed of three stages, at 98 ° C for 30 seconds, at 70 ° C for 20 seconds and 72 ° C for 7 minutes. He continued with 10 cycles composed of three stages, at 98 ° C for 20 seconds, at 70 ° C for 15 seconds and at 72 ° C for 7 minutes and, finally, a final cycle at 72 ° C for 15 minutes. After cooling on ice, the PCR product was treated with 2 U of Dpnl (Fermentas) for 6 hours at 37 ° C, 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 μΙ of 70% ethanol followed by cold centrifugation at maximum speed for 20 minutes. After complete drying of the precipitate in air, said precipitate is dissolved in 5 μΙ of sterile double-distilled water. With the product, 60 µΙ of chemically competent E. coli TG-1 cells were transformed by thermal shock. The culture was seeded on an agar-chocolate plate (Becton-Dickinson) with ampicillin (200 g / ml) and incubated at 37 ° C for 24 hours.

 The colonies obtained were analyzed by PCR with the primers

(SEQ ID NO: 12) 5 ^' -ctaggctggaagttctgttccaggggcccg-3 ^' and inverse (SEQ ID NO: 13) 5 ^' -ttttgaattc-ctctttatacttgctcacagcttcttt, and the PCR products were analyzed on 1% agarose gels 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 direct primers (SEQ ID NO: 14) 5 ^' - gaccaaagccatgacaaaaacg-3 ^' and inverse (SEQ ID NO: 15) 5 ^' -tcgacggcgctattcagatc - 3 ^' . In this case, 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 ° C with orbital shaking at 200 rpm. The cultures were cooled on ice, the cells were precipitated by centrifugation at 3,000 xg 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 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% sterile glycerol was added to an aliquot of the liquid culture of each colony and were stored at -80 ° C. The extracted plasmids were purified and sequenced, corroborating the expected sequence for plasmid pTOP-Tfam (SEQ ID NO: 19). In SEQ 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 coding for the Pre-Scission protease target comprises nucleotides 1.025-1 .048, the sequence encoding the third marker of Polyhistidine comprises nucleotides 1,049-1,078 and the sequence coding for 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 DNA (cDNA) obtained by retrotranscription with the M-MLV retrotranscriptase (Invitrogen) from blood lymphocyte messenger RNA extracted by trizol (Invitrogen). Primers for PCR were the forward primer (SEQ ID NO: 16) 5 ^{'- cacgatgacgatgacaag-gatgccggtgtgactgaa-3'} and reverse primer (SEQ ID NO: 17) ^{5'--atgacaactccgtcttcc} ctagctgcaccccat-3 ^', designed based to the cDNA 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 SEQ ID NO: 16 and nucleotides 19-35 of SEQ ID NO: 17) correspond to the sequence complementary to the site of hybridization with PCBP1. The resulting amplicon is 1,104 base pairs, which corresponds to the sequence of the PCBP1 cDNA from position 294 to 1 362 (1,068 base pairs, excluding the initiation ATG). The sequences of the 5 ^' ends (nucleotides 1-18 of SEQ ID NO: 16 and nucleotides 1-18 of SEQ ID NO: 17) and their complemenaries of the 3 ^' ends of the amplicon of 1,104 base pairs, hybridize with the vector pTOP at the sites ** - ##.

The PCR amplification of PCBP1 (insert) was performed in a final volume of 50 μΙ containing 10 μΙ of PrimeSTAR 5X buffer, mixture of 0.2 mM dNTPs each, 0.2 μΜ of each of the primers, 50 ng of cDNA and 1.25 U of PrimeSTAR HS DNA 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 ° C for 30 seconds; 40 cycles composed of three stages, at 98 ° C for 10 seconds, at 60 ° C for 30 seconds, and 72 ° C for 75 seconds and 1 final cycle at 72 ° C 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 μΙ / ml in TBE) and comparing it with the 1 Kb DNA molecular weight marker of GeneCraft. It was visualized with UV light and photographed on the documentation device of Bio-Vision gels. After cutting the band with a scalpel blade and purifying the products with the PCR product purification kit (Roche), 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 (SEQ 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 priming oligonucleotides of the first PCR), those that acted as primers in the CiPCR, and the sequences of the 5 ^' ends (contributed directly by the oligonucleotide primers to the amplicons of 1,104 base pairs of the first PCR) as termination signals of elongation For the CiPCR, a 25 μ 25 mixture containing 5 μΙ of 5X PrimeSTAR buffer, 0.2 mM dNTPs each, 250 ng of the PCBP1 amplicon, 100 ng of pTOP-Tfam plasmid and 1.25 U of PrimeSTAR was prepared HS DNA polymerase (Takara). The amplification conditions in the PTC200 thermal cycler were 1 cycle of denaturation at 95 ° C for 5 minutes, 5 cycles composed of four stages, at 95 ° C for 1 minute, at 65 ° C for 30 seconds, at 52 ° C for 30 minutes. seconds, and at 72 ° C for 7.5 minutes, 1 cycle consisting of four stages, at 98 ° C for 30 seconds, at 65 ° C for 20 seconds, at 52 ° C for 20 seconds and 72 ° C for 7.5 minutes, followed by 10 cycles composed of three stages, at 98 ° C for 20 seconds, at 80 ° C for 15 seconds and at 72 ° C for 7.5 minutes; and 1 final cycle at 72 ° C for 15 minutes. The CiPCR products were treated with 2 units of Dpnl for 6 hours at 37 ° C. After deactivating the enzyme for 20 minutes at 80 ° C, the products were precipitated with sodium acetate / ethanol, resuspended in 5 μΙ 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 g / 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 ° C as a source of bacteria for future crops. 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 - Merged with both polyhistidine and Glutathione S-Transferase labels (10His-GST-10His-PCBP1) immobilized or in solution

 3.2- Cut with the Pre-Scission 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 induction conditions of the expression of

PCBP1 in the plasmid pTOP, the bacterial stock strains E. coli BL21 were used with the plasmid pTOP-PCBP1 (SEQ ID NO: 18) that were stored in LB-glycerol at -80 ° C. An aliquot was seeded on a plate of LB-agar ampicillin (100 g / ml). One of the plate colonies was used fresh to inoculate 5 ml of LB in a 25 ml capacity tube that was grown for 6 hours at 37 ° C with stirring. This fresh culture was used to inoculate a flask containing 100 ml of LB with ampicillin (100 g / 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 aprotinin) corresponding, so that they all carry the same concentration of bacteria as the sample at time or induction. 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 blot was analyzed by Western blot. For this, the membranes were washed with PBS containing 0.05% Tween-20 (Sigma) and left blocking overnight at 4 ^o 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. The membrane was then 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 secondary anti-mouse antibody conjugated to horseradish peroxidase (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 LumigenTM PS-3 solution (Amersham) was used as a substrate, following the supplier's recommendations. 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 a calculation Approximate exposure time needed to obtain the signal with satisfactory intensity. The following films were exposed immediately after the calculation of the necessary exposure time. The result is shown in the Western 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 ° C previously seeded on a plate of LB-ampicillin agar (100 g / ml) This culture was incubated at 37 ° C and 200 rpm to reach 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 jars and centrifuged at 3,000 xg for 20 cold minutes. 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 xg for 20 minutes. The supernatant was incubated with 200 μΙ 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 two others. with lysis buffer containing 50 mM imidazole. Centrifugations between washings were done cold at 2,000 xg for 2 minutes. Then, 200 μΙ 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 10 μΙ aliquot was stored (containing 10His-GST-10His-PCBP1, Figure 4B lane 1), and the rest were added cold PBS until complete 1 mi and 150 μΙ of immobilized glutathione in balls (Amersham Biosciences), washed previously 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 μΙ of PreScission protease cut buffer was added to the precipitate of the balls, a 2 μΙ aliquot was stored (containing 10His-GST-10His-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 35 μΙ of protease cut buffer and 4 μΙ of Pre-Scission protease and the mixture was incubated with slow rotational agitation during 48 hours collecting aliquots of 65 μΙ every 12 hours with replacement of new cutting buffer (the Pre-Scission 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 10His-PCBP1). The protein immobilized in the balls was stored as such at 4 ° C for immediate use. The result can be seen in Figure 4B.

Claims

 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, d marker. a nucleotide sequence encoding a protease recognition amino acid sequence P,  and. a nucleotide sequence encoding an O marker, f. a nucleotide sequence encoding a protease recognition amino acid sequence Q,  g. a termination codon.  2. - The nucleotide sequence according to claim 1, 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, d marker. a nucleotide sequence encoding a protease recognition amino acid sequence P,  and. a nucleotide sequence encoding an O marker, f. a nucleotide sequence encoding a protease recognition amino acid sequence Q,  g. a termination codon.  3. - The nucleotide sequence according to any one of claims 1 or 2, 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 encoding a protease recognition amino acid sequence P, a nucleotide sequence encoding an O marker, a nucleotide sequence encoding a protease recognition amino acid sequence Q, a termination codon. 4. - The nucleotide sequence according to any of claims 1 to 3, characterized in that the markers M and O are the same. 5. - The nucleotide sequence according to any of claims 1 to 4, characterized in that the markers M, N and O are selected from the list comprising: polyhistidine, Glutathione S-transferase, avidin, streptavidin, hemagglutinin, VSV-G, HSVtk , FLAG, maltose binding protein, V5, myc and a fluorescent protein.  6. - The nucleotide sequence according to claim 5, wherein the markers M, N and O are selected from polyhistidine and Glutathione S-transferase.  7. - The nucleotide sequence according to claim 6, wherein the M and O markers are a polyhistidine and the N marker is the Glutathione S-transferase. 8. The nucleotide sequence according to claim 7, 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 one of claims 1 to 8, characterized in that the amino acid sequences P and Q of protease recognition are selected from the list comprising the targets of proteases: Pre-Scission, Enterokinase, Thrombin, Tev, HRV 3C, ULP1 and factor Xa.  10. - The nucleotide sequence according to claim 9, wherein 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 Enterokinase protease. 11. The nucleotide sequence according to any of claims 1 to 10, which further comprises an inducible promoter.  12. - The nucleotide sequence according to claim 1, wherein the inducible promoter is selected from the list comprising: arabinose inducible promoter, lactose inducible promoter or IPTG (isopropyl-pD-thiogalactoside), ecdysone inducible promoter, inducible promoter by tetracycline, 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 claim 12, wherein the promoter is arabinose inducible.  14. - The nucleotide sequence according to any of claims 1 to 13, which further comprises at least one origin of replication. 15. - The nucleotide sequence according to claim 14, which comprises the origin of replication of the plasmid pUC. 16. - The nucleotide sequence according to any of claims 1 to 15, which further comprises at least one selection gene.  17. - The nucleotide sequence according to claim 16, wherein the selection gene is chosen from the list comprising: an antibiotic resistance gene, a gene encoding a fluorescent protein, a gene encoding a luminescent protein, a gene that codes for beta-galactosidase and a gene that converts auxotrophic strains into prototrophs.  18. - The nucleotide sequence according to claim 17, wherein the selection gene is an antibiotic resistance gene. 19. The nucleotide sequence according to claim 18, 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 claim 19, wherein the selection gene is an ampicillin resistance gene.  21. - The nucleotide sequence according to any of claims 1 to 20, characterized in that it is SEQ ID NO: 1 or a bioequivalent variant.  22. - The nucleotide sequence according to any of claims 1 to 21, characterized in that it is a gene construct or is part of a gene construct.  23. - The nucleotide sequence according to claim 22, characterized in that it is a gene construct.  24. - The nucleotide sequence according to any of claims 22 or 23, characterized in that it is a vector or is part of a vector. 25. The nucleotide sequence according to claim 24, 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 claim 26, 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 claim 27, 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 eukaryotic cell, i ". Cultivate the cell in stage (i ') in the presence of the substances necessary for the expression of the protein encoded by the gene of interest cloned in step (h).  29. - The method according to claim 28, wherein in step (i ') the nucleotide sequence, gene construct or vector is introduced into a prokaryotic cell, preferably Escherichia coli.  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 claims 28 to 30, wherein the cultivation of step (i ") is done in the presence of arabinose.  32. - The method according to any of claims 28 to 31, further comprising the following step (j):  j. Purify the protein produced in step (i).  33. The method according to claim 32, characterized in that step (j) 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 ligand of step (j"). 34. The method according to claim 33, wherein the ligand immobilized on a solid support of step (j ") 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 claim 35, wherein the metal ion is nickel (I I) and / or cobalt (I I). 37. - The method according to any of claims 33 to 36, 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 displaces the protein by competition for ligand binding, treatment with a reducing agent, processing with a protease. 38. The method according to claim 37, wherein the protein is released from its ligand binding by processing with a protease. 39. The method according to any of claims 37 or 38, 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 claim 39, comprising the Pre-Scission 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 claim 44, wherein the vector is the nucleotide sequence SEQ ID NO: 1 or a bioequivalent variant.  46. - The kit according to any of claims 44 or 45, wherein the vector is the nucleotide sequence SEQ ID NO: 1.  47. - The kit according to any of claims 44 to 46, further comprising the instructions for carrying out the methods according to any of claims 26 to 43.  48. - The kit according to any of claims 44 to 47, which further comprises at least one protease.  49. - The kit according to claim 48, 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 claims 44 to 50, comprising a pair of direct and reverse primers of between 30 and 100 nucleotides, wherein the sequence of the direct primer coincides with the fragment of SEQ ID NO: 1 and whose end 3 ^' is the nucleotide at position 1.093 of SEQ ID NO: 1, and where the reverse primer sequence is complementary to SEQ ID NO: 1 so that its 3 ^' end is the nucleotide complementary to the nucleotide at position 1.094 of SEQ ID NO: 1.