WO2008032659A1 - Secretion signal peptide having improved efficiency, and method for production of protein by using the same - Google Patents

Abstract

Disclosed is a secretion signal peptide having a high secretion efficiency, which comprises an amino acid sequence having the substitution of a specific amino acid residue by another amino acid residue in the amino acid sequence for a secretion signal peptide derived from Saccharomyces cerevisiae α1-factor.

Description

 Specification

 Efficient secretion signal peptides and protein production methods using them

 Technical field

 [0001] The present invention relates to a secretory signal peptide and a protein production method using them.

 Background art

 [0002] Proteins include those that are synthesized inside a cell and then remain inside the cell and those that are released outside the cell. Proteins released outside the cell are generally called secreted proteins. In addition, there are proteins that have been synthesized in the cell and then penetrated into the cell membrane, endoplasmic reticulum membrane, etc., or partly inserted. These proteins are called membrane proteins.

 [0003] It is known that secreted proteins and membrane proteins have amino acid sequence characteristics different from those of other proteins. A relatively hydrophobic amino acid sequence is often found on the N-terminal side of these proteins. This amino acid coordination IJ is called "secretory signal peptide" (the sequence is called "secretory signal coordination 1]"! /). The importance of secretory signal peptides in the production of secreted proteins (extracellular secretion) and membrane proteins (membrane localization) eliminates secretory signal sequences from the amino acid sequences of secreted proteins and membrane proteins. Therefore, it is widely recognized that no extracellular secretion or membrane localization can be observed.

 [0004] In addition, even when a secretory signal peptide of a secreted protein or a membrane protein is replaced with a secretory signal peptide derived from another protein, in many cases, the same secretory signal peptide activity (secretion or localization to membrane) It is known that secretory proteins or membrane proteins substituted with secretion signal peptides derived from proteins of different species are also secreted extracellularly or localized in the membrane. . Thus, secretory signal peptides are widely used in fields such as molecular biology.

[0005] The secretory production of a protein using a secretory signal peptide has the following advantages: [0006] (1) In an intracellular expression system for proteins produced and accumulated in cells, the target protein accumulated in the cells may be degraded by proteolytic enzymes existing in the cells. Arise. On the other hand, in a protein secretion expression system using a secretory signal peptide, the target protein is secreted into the culture medium, so there is relatively little risk of degradation or the like.

[0007] (2) In a protein intracellular expression system, cells must be disrupted in order to purify a target protein, and all substances in the cell become contaminants. On the other hand, in a protein secretion expression system using a secretory signal peptide, the produced protein is secreted into the culture solution, so that cell disruption is unnecessary and the target protein is contained in the initial crude sample, that is, the culture solution. The target protein is relatively easy to purify.

 [0008] (3) In the intracellular expression system of a protein, the target protein is accumulated in a limited intracellular space, and therefore there is a risk of forming an aggregate at a high concentration. On the other hand, in a protein secretory expression system using a secretory signal peptide, the target protein is transferred to the culture medium, and as a result, it is diluted rather than produced intracellularly, and there is less possibility of such a problem. For this reason, the secretory expression system is suitable for mass production of proteins and is often used practically for the production of pharmaceuticals.

 [0009] (4) In a protein secretion expression system using a secretion signal peptide, the N-terminus of the target protein can be obtained as a natural protein. On the other hand, in an intracellular expression system in which the protein stays in the cell, it is often N-terminal force S-methionine or formylmethionine, which is not always the same as the natural protein, and biochemistry The activity may also be different from the natural type.

[0010] α 1 factor (α 1 mating factor) of budding yeast Saccharomyces cerevisiae is a secreted protein and has a secretory signal peptide. Specifically, the secretory signal peptide derived from the a1 factor is a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2. The secretory signal peptide consists of a pre-sequence consisting of the first to 19th amino acid sequences in SEQ ID NO: 2 and the 20th to 89th amino acids. And a prosequence consisting of a sequence. The pre-sequence and the pro-sequence are collectively referred to as pre-pro-binding IJ. In a narrow sense, a sequence containing a force prepro sequence whose pre sequence is a secretory signal sequence is often referred to as a secretory signal sequence.

 [001 1] The synthesized nascent α1 factor has a secretory signal peptide containing the above-mentioned preb mouth sequence on the heel end side. The α 1 factor having a secretory signal peptide synthesized intracellularly is cleaved by the endopeptidase in the process of translocation into the endoplasmic reticulum, and becomes an α ΐ factor precursor. The prosequence is then cleaved by the endoplasmic reticulum Kex2 endopeptidase at a site typified by a peptide bond between the 85th arginine and 86th glutamic acid of SEQ ID NO: 2. In addition, the Golgi Ste l 3 endopeptidase cleaves at the sites represented by peptide bonds on the C-terminal side of the 87th alanine and the C-terminal side of the 89th alanine of SEQ ID NO: 2. Through the processing, mature α1 factor will be synthesized. The synthesized mature α1 factor is secreted extracellularly.

 [0012] Examples of secretory signal peptides derived from Saccharomyces cerevisiae proteins include secretory signal peptides derived from proteins such as α-factor receptor, invertase, and phosphatase, in addition to the above-mentioned secretory signal peptides derived from α1 factor. Is known and used so far. However, these secretory signal peptides have a common force in terms of their ability to secrete proteins and peptides connected to their C-terminal side (in the case of membrane proteins, they are produced in the membrane). The ability to transport and secrete a large amount of proteins and peptides to the outside of the cell and to secrete them. (In the case of membrane proteins, In fact, the α1 factor-derived secretory signal peptide is very often used because of its high secretion efficiency, and there are many achievements related to the secretory production of various proteins. It has been known.

[0013] Examples of proteins secreted and produced using a secretory signal peptide derived from factor a 1 include, for example, invertase (Non-patent document 1), epidermal growth factor (Non-patent document 2), interferon- _α 1 ( Non-patent document 3), 13-endorphin (non-patent document 4), anti-CD33 single chain antibody (non-patent document 5), phytase (non-patent document 6) and the like are known. A 1 factor It is known that the secretory signal peptide derived can function as a secretory signal peptide not only in Saccharomyces cerevisiae but also in other yeast species and can secrete proteins (Non-patent Document 7). In addition, the secretory signal peptide derived from αΐ factor is also used in an expression system (Invitrogen) using methanol yeast Pichia pastoris. Thus, the secretory signal peptide derived from α1 factor can be widely applied to secrete and express a protein.

 [0014] By the way, attempts have been made to improve the secretory efficiency of linked proteins by modifying pre-sequences and pro-sequences derived from secreted proteins or membrane proteins.

 Patent Document 1 discloses an invention relating to modification of a pre-sequence. In the invention described in Patent Document 1, most of the natural signal arrangement IJ (signal sequence in a narrow sense) of egg white lysozyme is substituted with leucine, which is a hydrophobic amino acid. According to Patent Document 1, when a gene encoding a fusion protein in which a DNA sequence encoding the modified signal sequence and a DNA sequence encoding human lysozyme are linked is expressed in Saccharomyces cerevisiae, the natural signal sequence The amount of secreted protein is increased as compared with the case of using. However, the increase in secretion volume has only been about 2.5 times.

 On the other hand, Patent Document 2 discloses an invention relating to modification of a prosequence. Patent Literature

 2 includes one glycosylation site (eg, the 23rd asparagine in SEQ ID NO: 2) of the three pro sequences derived from α1 factor and its glycosylation recognition sequence IJ (23rd A gene encoding a pro-sequence that leaves a portion encoding Asnolagin followed by 2 amino acids) and from which part or all of the open-ended sequence on the C-terminal side has been deleted, and its 3 ′ end It was disclosed that a gene encoding a fusion protein was constructed in which a gene coding for a Kex2 cleavage site was linked to the side, and a gene encoding an insulin-like substance protein was linked to the 3 ′ end. Yes. According to the description in Patent Document 2, when the gene encoding the fusion protein is expressed in Saccharomyces cerevisiae, the amount of the protein secreted increases as compared with the case where a natural prosequence is used. However, the increase in secretion is less than three times, and it is difficult to say that it has improved dramatically.

[0017] In recent years, as well as the production of protein and industrial enzymes as pharmaceuticals, as seen in the "Protein 3000" project of the Ministry of Education, Culture, Sports, Science and Technology, protein testing for elucidating biochemical phenomena Development of a protein expression system capable of producing a desired protein simply and in large quantities is strongly demanded as a means for providing a material. Therefore, there is a need for further improvement in the secretory expression system of proteins in terms of improving the secretory efficiency.

 In addition, when a membrane protein is synthesized, it is generally known to use a mechanism common to secreted proteins, such as passage through the endoplasmic reticulum membrane by a signal sequence. Therefore, it is considered that the secretory signal peptide described above can also be applied to efficiently produce a membrane protein.

Patent Document 1: Japanese Patent No. 2564536

Patent Document 2: Japanese Patent No. 2793215

 ^ Patents l: Emr, ¾.D., Schekman, R., rlessel, し., Thorner, j., “Proceedings of the National Academy of Sciences of the United States of America J, 1983, 80th , .7080-7084

Non-Patent Document 2: Brake, AJ, Merryweather, JP, Coit, DG, Heberlin, UA, Masiarz, FR, Mullenback, GT, Urdea, MS, Valenzuela, P., Barr, PJ, `` Proceedings of the National Academy of Sciences of the United States of America J, 1984, Vol. 81, p.4642-4646

Non-Patent Document 3: Singh, A., Lugovoy, J., ohr, W., Perry, L.J., "Nucleic Acids Research", 1984, Vol. 12, No. 23, p.8927-8938

Non-Patent Document 4: Bitter, GA, Chen,.., Banks, AR, Lai, Ρ_Η ·, “Proceedings of the National Academy of Sciences of the United States of America J, 1984, —81, p.5330- 5334

Non-Patent Document 5: Emberson, LM, Trivett, AJ, Blower, PJ, Nicholls, PJ, “JOURNA L OF IMMUNOLOGICAL METHODS], 2005, Vol. 305, No. 2, ρ · 135- 151 Non-Patent Document 6: Xiong, AS, Yao, QH, Peng, RH, Han, P 丄., Cheng, ZM, Li, Y., jOURNAL OF APPLIED MICROBIOLOGYJ, 2005, Vol. 98, No. 2, ρ · 418_428 ^ Patents: Kalandadze, A., alleno, M., roncerrada, and Strominger, JL, Wucherpfennig, .W., “THE JOURNAL OF BIOLOGICAL CHEMISTRY”, 1996, Vol. 27, No. 33, .20156-20162 Disclosure of the invention

 Problems to be solved by the invention

 [0019] As described above, it is an extremely important issue to further improve the production efficiency of secreted proteins and membrane proteins.

 [0020] Therefore, an object of the present invention is to identify and provide a secretory signal peptide having higher secretory efficiency than, for example, conventionally used secretory signal peptides.

 Means for solving the problem

 [0021] As a result of diligent research to solve the above problems, a secretion signal consisting of an amino acid sequence in which a specific amino acid residue is replaced with another amino acid in the secretion signal peptide derived from α1 factor of Saccharomyces cerevisiae Peptide power We found that the secretory production amount of the protein linked to the C-terminal side can be improved at least 3 times compared to the secretion signal peptide derived from the natural α1 factor, and the present invention was completed. It came to do.

 [0022] The present invention includes the following.

 [0023] (1) A secretion signal peptide of the following (a) or (b):

 (A) In the amino acid sequence described in SEQ ID NO: 2, the 19th alanine, the 20th alanine, the 21st proline, the 22nd valine, the 23rd asparagine and the 24th Secretory signal peptide consisting of an amino acid sequence in which at least one amino acid selected from the group consisting of threonine of the eye is replaced with another amino acid

 (b) In the amino acid sequence of the secretion signal peptide of (a) above, an amino acid sequence in which one or several amino acids are deleted, substituted or added at positions other than the 19th to 24th amino acids. A secretory signal peptide that improves the secretory production of a protein linked to the C-terminal side by 3 times or more compared to a secretory signal peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2

 (2) The secretory signal peptide according to (1), wherein the 19th alanine is replaced with another amino acid! /.

 [0025] (3) The secretion signal peptide according to (1) or (2), wherein the 19th alanin force S-parin is substituted.

[0026] (4) The 20th alanine is substituted with another amino acid, (1) ) The secretory signal peptide described.

[0027] (5) The secretory signal peptide according to (1) or (4), wherein the 20th alanine is substituted with threonine.

[0028] (6) The secretory signal peptide according to (1), wherein the 21st proline is substituted with another amino acid.

[0029] (7) The secretory signal peptide according to (1) or (6), wherein the 21st proline is substituted with leucine.

[0030] (8) The secretion signal peptide according to (1), wherein the 22nd palin is substituted with another amino acid.

[0031] (9) The secretory signal peptide according to (1) or (8), wherein the 22nd valine is substituted with phenylalanin or alanine! /.

[0032] (10) The secretory signal peptide according to (1), wherein the 23rd asparagine is substituted with another amino acid.

[0033] (11) The 23rd asparagine is substituted with tyrosine,

The secretion signal peptide according to 1) or (10).

[0034] (12) The 24th threonine is substituted with another amino acid,

 (1) The secretion signal peptide according to the above.

[0035] (13) The secretory signal peptide according to (1) or (12), wherein the 24th threonine is substituted with alanine.

[0036] (14) A DNA encoding the secretory signal peptide according to any one of (1) to (; 13).

[0037] (15) A DNA fragment comprising the DNA according to (14).

[0038] (16) A recombinant vector comprising the DNA according to (14).

 [0039] (17) A fusion protein obtained by linking the secretion signal peptide according to 1 and a foreign protein according to any one of (1) to (; 13).

[0040] (18) The fusion protein according to (17), wherein the foreign protein is linked to the C-terminal side of the secretory signal peptide!

[0041] (19) DNA encoding the fusion protein according to (17) or (18).

[0042] (20) A DNA fragment comprising the DNA according to (19). [0043] (21) A recombinant vector comprising the DNA according to (19).

 [0044] (22) A transformant having the DNA fragment according to (20) or the recombinant vector according to (21)

Yes

 [0045] (23) The transformant according to (22), wherein the host is yeast.

 [0046] (24) The transformant according to (23), wherein the yeast is Saccharomyces cerevisiae.

 [0047] (25) A method for producing a protein, characterized by culturing the transformant according to any one of (22) to (24) and allowing the foreign protein to be secreted or expressed in a membrane.

 The invention's effect

 [0048] According to the present invention, there is provided a secretory signal peptide exhibiting high / low secretion efficiency of linked proteins when cells of various species such as Saccharomyces cerevisiae are used as hosts. According to the present invention, the protein secretion productivity is improved, and the means for supplying protein as an industrial or research sample is greatly improved.

 [0049] This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2006-249721, which is the basis of the priority of the present application.

 Brief Description of Drawings

 [0050] [FIG. 1] FIG. 1 shows luciferase (CLuc) in each mutant and wild-type culture supernatant.

V shows the relative emission intensity.

 [FIG. 2] FIG. 2 shows luciferase (CLuc) in each mutant and wild-type culture supernatant.

V shows the luminescence value (relative value) per microbial mass (OD600).

 BEST MODE FOR CARRYING OUT THE INVENTION

[0051] Hereinafter, the present invention will be described in detail.

 [0052] The secretory signal peptide according to the present invention is a variant of the secretory signal peptide derived from α1 factor of Saccharomyces cerevisiae. Specifically, it is the following secretory signal peptide (a) or (b).

[0053] (a) In the amino acid sequence described in SEQ ID NO: 2, the 19th alanine, the 20th alanine, the 21st proline, the 22nd valine, the 23rd asparagine and the 24th At least one amino acid selected from the group consisting of threonine of the eye is substituted for other amino acids. A secretory signal peptide consisting of a converted amino acid sequence;

 (b) In the amino acid sequence of the secretion signal peptide of (a) above, an amino acid sequence in which one or several amino acids are deleted, substituted or added at positions other than the 19th to 24th amino acids. A secretory signal peptide that improves the secretory production of a protein linked to the C-terminal side by 3 times or more compared to a secretory signal peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2

 [0054] The peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 is a secretory signal peptide derived from Saccharomyces cerevisiae α1 factor. In the amino acid sequence shown in SEQ ID NO: 2, the first to 19th amino acid sequences are pre-sequences, while the 20th and the 89th amino acid sequences are pro-sequences. The base sequence shown in SEQ ID NO: 1 is a gene (cDNA) encoding a secretory signal peptide derived from Saccharomyces cerevisiae α1 factor.

 [0055] The secretion signal peptide described in the above (a) is the 19th alanine, the 20th alanine, the 21st in the a1 factor-derived secretion signal peptide (amino acid sequence IJ described in SEQ ID NO: 2). A secretory signal peptide comprising an amino acid sequence in which at least one amino acid selected from the group consisting of proline, 22nd valine, 23rd asparagine and 24th threonine is substituted with another amino acid. By this amino acid substitution, the secreted production amount of the protein linked to the C-terminal side in a host such as yeast as compared with the secretory signal peptide derived from the wild type α1 factor (that is, the peptide consisting of the amino acid sequence described in SEQ ID NO: 2) Can be improved by 3 times or more (for example, 3 to 20 times, preferably 5 to 20 times). Alternatively, by this amino acid substitution, in a host such as yeast, compared to the secretory signal peptide derived from wild type α1 factor (that is, the peptide consisting of the amino acid sequence described in SEQ ID NO: 2), it was linked to the C-terminal side. It is possible to improve the localization of the protein membrane (cell membrane, endoplasmic reticulum membrane, etc.).

 [0056] The substitution at the amino acid position described above may be single or a combination of two or more.

[0057] Here, the other amino acids are! / Other than the specific amino acids of the secretion signal peptide derived from the wild-type α1 factor at each position described above, and may be misplaced amino acids! /. Ni! / The following amino acids are preferably substituted.

[0058] (1) 19th alanine: substitution with valine, leucine or isoleucine

 (2) 20th alanine: substitution with threonine or serine

 (3) 21st proline: substitution with leucine, norline or isoleucine

 (4) 22nd valine: substitution with phenylalanin or alanine

 (5) 23rd asparagine: substitution to tyrosine or phenylalanine

 (6) 24th threonine: substitution with alanine or parin

 [0059] On the other hand, the secretion signal peptide described in (b) above is further one or several (eg, for example) at positions other than the 19th to 24th amino acids in the secretion signal peptide described in (a). 1 to 10, preferably 1 to 5, particularly preferably 1 to 3 amino acids), which is composed of an amino acid sequence deleted, substituted or added, and compared with a secretory signal peptide derived from wild type α1 factor Thus, the secretory production of the protein linked to the C-terminal side can be improved by 3 times or more. Examples of positions other than the 19th to 24th amino acids include the 18th and 25th amino acids.

 [0060] Furthermore, the secretory signal peptide according to the present invention retains a predetermined amino acid substitution at the 19th to 24th amino acid positions of the secretory signal peptide described in (a) above. It has at least 70% or more, 80% or more, 90% or more, preferably 95% or more amino acid sequence identity with the amino acid sequence of the described secretory signal peptide, and compared with the secretory signal peptide derived from wild-type α1 factor Also included are peptides that can increase the secretory production of proteins linked to the C-terminal side by a factor of 3 or more.

 [0061] The secretory signal peptide according to the present invention can be made into a fusion protein linked to a foreign protein. Here, the foreign protein means a protein exogenous to the secretory signal peptide according to the present invention. For the foreign protein, the peptide is also ρ¾.

Here, the foreign protein is not particularly limited, and may be any naturally occurring secreted protein, membrane protein, or other non-secretory protein. However, when a non-secretory protein is linked to a secretory signal peptide, there are many cases where no secretion is observed. Therefore, in the present invention, non-secretory protein A protein that can be secreted in quality is called a foreign protein. When a secreted protein or membrane protein is linked, it is preferable to link the mature protein obtained by removing / excluding the secretory signal peptide naturally present in the protein to the secretory signal peptide according to the present invention. For example, the secretory signal peptide portion of a secreted protein or membrane protein can be determined by subjecting the mature protein to N-terminal amino acid analysis and comparing it with the deduced amino acid sequence obtained from the base sequence of cDNA. Alternatively, it is estimated by a signal peptide prediction program such as SignalP3.0 (http://www.cbs.dtu.dk/services/SignalP/ and Nielsen H., (1997) Protein Engineering, 10: 1-6). It is also possible.

 [0063] Examples of foreign proteins include various hormones (such as insulin) of proteins or peptides, erythropoietin, cytodynamic force (such as interferon), and various enzymes (such as invertase and amylase). Alternatively, proteins with unknown functions discovered by large-scale genome analysis in recent years can also be targeted.

 [0064] The position at which the foreign protein is linked to the secretory signal peptide according to the present invention can be appropriately selected so that the secretory signal peptide according to the present invention and the foreign protein have their respective functions or activities. It is preferable that a foreign protein is linked to the C-terminal side of the secretory signal peptide according to the invention.

 [0065] The DNA according to the present invention is DNA encoding the secretory signal peptide according to the present invention or DNA encoding the above-mentioned fusion protein (hereinafter referred to as "DNA encoding the fusion protein according to the present invention"). . In particular, by introducing a DNA fragment containing a DNA encoding the fusion protein according to the present invention or a recombinant vector into a host, the foreign protein in the fusion protein can be secreted or expressed on a membrane. In addition, the DNA encoding the secretory signal peptide according to the present invention is preliminarily incorporated into an expression vector (a vector having a promoter and the like and used mainly for protein production) to facilitate the construction of a plasmid for the production of foreign proteins. It is possible to

 [0066] Hereinafter, production of DNA encoding the fusion protein according to the present invention, or a DNA fragment or recombinant vector containing the DNA will be described.

[0067] First, in preparation of DNA encoding the fusion protein according to the present invention, the analysis according to the present invention is performed. Prepare DNA encoding the signal peptide and DNA encoding the foreign protein. In preparation of a DNA encoding a secretory signal peptide according to the present invention, for example, a genomic DNA of Saccharomyces cerevisiae is used as a saddle type, and primers complementary to the nucleotide sequences at both ends of the region encoding the α1 factor-derived secretory signal peptide The wild type α1-factor-derived secretory signal peptide coding region is amplified by PCR using. Next, the secreted signal peptide according to the present invention is encoded by a known mutagenesis method (for example, Kunkel method or PCR using a synthetic oligonucleotide as a primer for mutagenesis) on the amplified PCR product. Can make DNA. Alternatively, DNA encoding the secretory signal peptide according to the present invention can be chemically synthesized.

 [0068] On the other hand, the DNA encoding the desired foreign protein is a genomic DNA of the organism from which the foreign protein is derived, cD mRNA, etc., and a primer complementary to the nucleotide sequences at both ends of the foreign protein coding region. It can be obtained by amplification using the PCR used. Alternatively, when DNA encoding a foreign protein has already been cloned, it can be obtained, for example, by cutting it out from the vector with a restriction enzyme from the vector containing the DNA. Alternatively, DNA encoding a foreign protein can be chemically synthesized.

 [0069] The DNA encoding the secretory signal peptide according to the present invention and the DNA encoding the foreign protein are linked so that the codon frames match. As described above, the positional relationship between the DNA encoding the secretory signal peptide according to the present invention and the DNA encoding the foreign protein can be appropriately selected so that the expressed fusion protein functions. Strength It is preferable that the DNA encoding the foreign protein is operably linked to the 3 ′ end of the DNA encoding the secretory signal peptide according to the present invention. As a means for ligation, an enzyme (DNA ligase) reaction is generally used. Alternatively, double-stranded DNA having sequences already linked may be chemically synthesized.

[0070] The recombinant vector according to the present invention can be obtained by inserting a DNA encoding the fusion protein according to the present invention into an appropriate vector. The vector to be used is not particularly limited as long as it can be replicated in the host, and examples thereof include a plasmid, a shuttle vector, and a neuroper plasmid. When a shuttle vector is used, for example, a DNA distribution that allows the vector to replicate autonomously in Escherichia coli. 歹 By including IJ, it can be maintained and replicated in cells both in the host used for protein expression and in E. coli. Further, if the vector itself does not have replication ability, it may be a DNA fragment that can be replicated by inserting it into the host chromosome.

 [0071] In the recombinant vector according to the present invention, the promoter having transcription initiation activity in the host is functionally linked to the 5 'end of the DNA encoding the fusion protein according to the present invention. . That is, the recombinant vector according to the present invention is inserted so that a promoter suitable for the host is functionally linked to the 5 ′ end of the DNA encoding the fusion protein according to the present invention. Is preferred. The promoter may be inducible or non-inducible (constitutive). From the viewpoint of protein secretion productivity, a promoter that is recognized to have a strong transcription initiation activity in the host to be used, for example, a promoter derived from a gene encoding a Saccharomyces cerevisiae glycolytic enzyme is desirable. For example, examples of such a promoter include a promoter derived from a TDH3 (glyceroanolaldehyde triphosphate dehydrogenase) gene.

 [0072] Alternatively, in the production of the recombinant vector according to the present invention, the promoter, the DNA encoding the differentiation signal peptide according to the present invention, and the DNA encoding the foreign protein are separately inserted and ligated. Can also be constructed.

 [0073] On the other hand, in the DNA fragment according to the present invention, in accordance with the production of the recombinant vector according to the present invention described above, the promoter that has transcription initiation activity in relation to the host is a fusion protein according to the present invention. It is preferably functionally linked to the 5 ′ end of the DNA encoding the.

 [0074] It should be noted that, in accordance with the preparation of the above-described recombinant vector or DNA fragment according to the present invention, for example, the secretory signal peptide according to the present invention is encoded as a DNA fragment or recombinant vector for producing a foreign protein. A DNA fragment or recombinant vector containing the DNA to be prepared can also be produced. Next, DNA encoding a foreign protein to be secreted or membrane expressed is appropriately inserted into the DNA fragment or recombinant vector.

[0075] Furthermore, the DNA fragment or recombinant vector according to the present invention containing the DNA encoding the fusion protein according to the present invention (hereinafter referred to as "the recombinant vector according to the present invention") is introduced into the host. To produce a transformant. The host is not particularly limited 1, e.g., true fungi and animal cells containing various yeasts. The yeast may be any yeast, for example, Saccharomyces 'cereviche, Schizosaccharomyces pombe, Pichia' pastris, Candida albicans, Hansenula polymorpha (Hansenula polymorpha). Saccharomyces cerevisiae is particularly preferred from the viewpoint that transformation methods and gene manipulation techniques have been established. It has also been confirmed that the Saccharomyces cerevisiae α1 factor-derived secretory signal peptide can function in different yeasts other than Saccharomyces cerevisiae (Non-patent Document 7). Other fungi include the genus Aspergillus, the genus Penicillium, the genus Trichoderma, the genus Rhizopus j¾, the genus Mucor and the like.

 [0076] The method for introducing the recombinant vector according to the present invention into the host is not particularly limited as long as it is a method for introducing DNA into the host. For example, electroporation (elect mouth position method), spheroplast method, Examples include the lithium acetate method. Alternatively, it may be a transformation method of yeast or the like such as substitution or insertion into a vector such as a Yip system or a chromosome. Furthermore, the method for introducing the recombinant vector according to the present invention into a true fungus or animal cell containing yeast or the like may be described in a general academic book, academic paper, or the like. ! /

 [0077] Further, by adding a DNA fragment according to the present invention in which a promoter and a gene encoding the fusion protein according to the present invention are linked, DNA having homology with the host chromosomal DNA sequence is added to both ends thereof, Instead of using a vector, it can be incorporated into the chromosomal DNA of the host cell by the homologous recombination function inherent to the host. The introduction of the DNA fragment into the host can be performed according to the above-described method for introducing the recombinant vector according to the present invention.

[0078] Confirmation of whether or not the recombinant vector according to the present invention has been incorporated into a host can be performed by PCR, Southern hybridization, Northern hybridization, or the like. For example, DNA is prepared from the transformant, PCR is performed by designing DNA-specific primers. Thereafter, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with bromide zyme, SYBR (registered trademark) Green solution, etc., and the amplified product is detected as a band. Confirm that the product has been transformed. In addition, using a primer previously labeled with a fluorescent dye, etc. CR can be used to detect the amplification product. Further, a method may be employed in which the amplification product is bound to a solid phase such as a microplate and the amplification product is confirmed by fluorescence, enzyme reaction, or the like.

 [0079] Next! /, The obtained transformant is cultured under conditions capable of growth. V, in the case of deviation, it is desirable to set the culture conditions of the transformant in consideration of the characteristics of the host and the stability of the desired foreign protein. For example, as shown in the present example, when the budding yeast Saccharomyces cerevisiae is used as a host to secrete and express luciferase (corresponding to a foreign protein), the yeast grows and the luciferase is not inactivated. The culture temperature is set to, for example, 4 to 37 ° C, preferably 20 to 30 ° C. The pH of the medium may be set to 3 · 5 to 6 · 5, preferably 5 · 5 to 6 · 0, for example. The culture time is, for example, 1 to 120 hours, preferably 1 to 24 hours in the logarithmic growth phase.

 [0080] When the foreign protein becomes a secreted protein, the secretory production amount of the foreign protein may be evaluated by any method as long as the production amount of the foreign protein can be specifically measured. The culture supernatant obtained by subjecting the solution to centrifugation can be measured using the enzyme activity, physiological activity, etc. of the foreign protein as indicators. In addition, the amount of secretion of foreign protein can be measured by a general immunological technique such as Western plotting or ELISA using an antibody specific to the target foreign protein. Alternatively, a method using a fluorescence microscope or flow cytometry may be used.

 [0081] On the other hand, when the foreign protein is a membrane protein, after separating the transformant, the membrane fraction is separated, so that the cell membrane or the like can be isolated according to the above-described method for measuring the amount of secreted protein produced. The amount of localization on the membrane can be measured. Alternatively, the amount of localization on the cell membrane may be measured by subjecting the transformant to flow cytometry using an antibody specific for the foreign protein as it is.

[0082] In this way, the desired foreign protein secreted and produced can be obtained from a culture supernatant by a conventional protein purification method such as ammonium sulfate salting out, ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography. It can be purified by high performance liquid chromatography, affinity chromatography, isoelectric focusing, polyacrylamide gel electrophoresis, etc. [0083] As described above, according to the secretory signal peptide of the present invention, a foreign protein can be efficiently secreted and produced in a host. The protein expression system using the secretory signal peptide according to the present invention is a secretory protein expression system. The secretory protein expression system has the advantage that it is relatively easy to purify the initial crude sample, that is, the culture medium with less contaminants other than the desired protein than the expression system that accumulates the desired protein in the cell. Have

 Example

 [0084] Hereinafter, the ability to explain the present invention in more detail with reference to examples. The technical scope of the present invention is not limited to these examples.

 [0085] In this example, as a foreign protein secreted by the improved secretion function of the secretory signal peptide according to the present invention, luciferase (hereinafter referred to as "CLuc") derived from Cypridina noc tiluca Used).

 [0086] On the other hand, Saccharomyces cerevisiae was used as the host.

 [0087] The expression plasmid pCLuRA-TDH3 is an expression vector that allows CLuc to be expressed in Saccharomyces cerevisiae. International Publication No. 2006/132350 (International Application PCT / JP2006 / 311597; Japanese Patent Application 2005- No. 169768 is the basis of priority).

 [0088] This plasmid pCLuRA-TDH3 is a secreted signal peptide derived from wild type α1 factor (amino acid sequence: SEQ ID NO: 2) and a mature protein of CLuc (1st to 18th in the amino acid sequence of CLuc shown in SEQ ID NO: 3). And a gene encoding a fusion protein (hereinafter referred to as “a CLuc gene”) with the amino acid sequence (the amino acid sequence excluding the IUC corresponding to the CLuc secretion signal peptide).

 [0089] Furthermore, the plasmid pCLuRA_TDH3 has a saccharomyces. Cerevisiae TDH3 (systematic gene name: YGR192C) gene promoter operably incorporated upstream (5 ') of the a CLuc gene. Thus, when this promoter is activated, a CLuc gene is expressed, and CLuc is secreted extracellularly by the function of the α1 factor-derived secretory signal peptide.

[0090] In addition, plasmid pCLuRA-TDH3 is essential for replication of plasmids that function in E. coli. Contains essential DNA sequences and ampicillin resistance gene. This plasmid is therefore a shuttle vector that is replicated and maintained in both Saccharomyces cerevisiae and E. coli.

 [0091] The base sequence shown in SEQ ID NO: 4 is a partial base sequence of the plasmid pCLuRA-TDH3, which shows the TDH3 gene promoter, a CLuc gene, and the 5 'and 3' base sequences thereof.

 [Example 1] Secretory expression of CLuc using a mutant α1-factor-derived secretory signal peptide (corresponding to the secretory signal peptide according to the present invention)

 1-1. Construction of a mutant α1 factor-derived secretory signal peptide gene library In the plasmid pCLuRA-TDH3, the region encoding the α1 factor-derived secretory signal peptide (from the 701st position in the nucleotide sequence shown in SEQ ID NO: 4) Error Prone PCR (error-prone PCR) was performed targeting the region corresponding to the 955th base). In this error prone PCR, the following oligo DNA primers were used.

[0093] sgl-f: CACCAAGAACTTAGTTTCGAGGG (IJ No. 5) The composition of this Error Prone PCR reaction solution was as follows: Taq DNA polymerase (Roche, 5 unit / μ 1) 1 μ \; lOxPCR Duffer without magnesium ion 10 μ1; Deoxynucleotide mixed solution for Error Prone PCR 10 μ1; 25 mM magnesium chloride 28 μ1; 5 mM manganese chloride 5.0 1; Plasmid pCLuRA-TDH3 solution (15θΓ¾ / 1) 1 ^ 1; sgl-i (SEQ ID NO: 5 XlOpmol / a 1) 3 μ1; sg2_r (SEQ ID NO: 6) (10 pmol / 〃 1) 3 1; Sterile water 39 μ1.

 [0094] The composition of the above-mentioned error-prone PCR-use doxynucleotide mixed solution was as follows: lOOmM dCTP 100 μ 1; lOOmM dTTP 100 μ 1; lOOmM dGTP 20 μ 1; 100 mM dATP 20 a 1; sterilized water 760 μl.

 [0095] Error Prone PCR was performed at 94 ° C for 1 minute (denaturation), 45 ° C for 1 minute (annealing), and 72 ° C for 1 minute (extension) in 30 cycles. The DNA fragment obtained by Error Prone PCR corresponds to the region between the 678th to 990th bases in the base sequence shown in SEQ ID NO: 4.

[0096] Next! /, Use 1% agarose to remove the entire PCR reaction solution obtained by Error Prone PCR. As a result of electrophoresis, a DNA fragment of about 300 bp was confirmed. This DNA fragment was purified by ethanol precipitation with Sigma Gen Elute ™ MINUS EtBr SPIN COLUMNS and dissolved in 10–1 TE buffer (10 mM Tris-HC1 ImM EDTA, ρΗ8.0). did. “The DNA solution Aj contains DNA molecules with random point mutations introduced by Error Prone PCR.

[0097] In the base sequence shown in SEQ ID NO: 4, the region between the 460th to 700th bases was amplified by PCR. The following oligo DNA primers were used in this PCR:

[0098] SQ-GPD 1-F0: CATGTATCTATCTCATTTTCTTAC (IJ No. 7) The composition of this PCR reaction solution was as follows: KOD plus DNA polymerase (Toyobo) 0.4 μ 1; 10x KOD plus buffer 2 μ \; 2 mM each dNTP mixture 2 μ \; 25 mM magnesium sulfate 0.8 1; SQ-GPD 1-F0 (SEQ ID NO: 7) (10 01 ₀ 1/1) 0.6 1; sgl_r (SEQ ID NO: 8) (10 pmol / 1) 0.6 1; Plasmid pCLuRA_TDH3 solution (lng / 1) 1 μ 1; Sterile water 12.6

[0099] PCR was performed at 94 ° C for 2 minutes (inactivation of anti-polymerase antibody) for one cycle, and at 94 ° C for 15 seconds (denaturation), at 50 ° C for 30 seconds (annealing), and at 68 ° C. A cycle of 1 minute (extension) was performed in 30 cycles. The entire PCR reaction solution obtained by this PCR was electrophoresed with 1% agarose. As a result, an approximately 250 bp DNA fragment was confirmed. This DNA fragment was purified by Sigma GenElute ™ MINUS EtBr SPIN COLUMNS and ethanol precipitation and dissolved in 10-1 TE buffer to obtain “DNA solution B”.

 [0100] Furthermore, in the base sequence shown in SEQ ID NO: 4, the region between the 968th to 1663th bases was amplified by PCR. The following oligo DNA primers were used in this PCR:

[0101] sg2-f: CAGGACTGTCCTTACGAACCTGA (IJ number 9)

SQ-CLuc-CRl: TGGACAACCGTCAAACTCCTGGTTGATCTT (IJ No. 10) The composition of this PCR reaction solution was as follows: KOD plus DNA polymerase 0.4 ^ 1; 10x KOD plus buffer 2 μ \; 2mM each dNTP mixture 2 μ \; 25 mM magnesium sulfate 0.8 a 1; sg2-f (SEQ ID NO: 9) (10 pmol / μ 1) 0.6 μ 1; SQ-CLuc-CRl (SEQ ID NO: 10) (lOpmo 1 / a 1) 0.6 a 1; plasmid pCLuRA-TDH3 (lng / ^ 1) 1 ^ 1; sterilized water 12.6 μ1.

[0102] PCR was performed at 94 ° C for 2 minutes (inactivation of anti-polymerase antibody) for 1 cycle, and at 94 ° C for 15 seconds (denaturation), at 50 ° C for 30 seconds (annealing), and at 68 ° C A cycle of 1 minute (extension) was performed in 30 cycles. As a result of electrophoresis of the entire PCR reaction solution obtained by this PCR with 1% agarose, a DNA fragment of about 700 bp was confirmed. This DNA fragment was purified by Sigma GenElute ™ MINUS EtBr SPIN COLUMNS and ethanol precipitation, and dissolved in 10-1 TE buffer to obtain “DNA solute night C”.

 [0103] Next, PCR was performed using the obtained mixture of DNA solution A, DNA solution B, and DNA solution C as a bowl. The DNA fragment obtained by PCR corresponds to the region between the 460th force and the 1663th base in the nucleotide sequence shown in SEQ ID NO: 4. However, since the DNA solution A obtained by Erro r Prone PCR is used as part of the saddle type, a point mutation was introduced into the region encoding the α 1 factor-derived secretory signal peptide. Contains DNA molecules.

 [0104] The composition of this PCR reaction solution was as follows: KOD plus DNA polymerase 1 μ 1;

10χ KOD plus buffer 5 ^ 1; 2 mM each dNTP mixture 5 ^ 1; 25 mM magnesium sulfate 2 μΐ; SQ-GPD1-F0 (SEQ ID NO: 7) (10 01 ₀ 1/1) 1.5 1; SQ-CLuc-CRl ( SEQ ID NO: 10) (lOpmol / 1) 1.5 1; DNA solution A 3 μ \; DNA solution Β 1 μ 1; DNA solution C 1 μ 1; Sterile water 29

[0105] PCR was performed at 94 ° C for 2 minutes (inactivation of anti-polymerase antibody) for 1 cycle, and at 94 ° C for 15 seconds (degenerative), 50 ° C for 30 seconds (annealing), and 68 ° C. A cycle of 1 minute 20 seconds (extension) was performed in 30 cycles. As a result of electrophoresis of a portion of the PCR reaction solution obtained by this PCR reaction with 1% agarose, a DNA fragment of about 1.2 kbp was confirmed. The remaining PCR reaction solution was purified by Sigma GenElute ™ PCR Clean-Up Kit and ethanol precipitation, dissolved in 50 1 TE buffer, and used as “DNA Purification Night D”.

[0106] On the other hand, in the base sequence of plasmid pCLuRA-TDH3, the base sequence shown in SEQ ID NO: 4 is deleted! /, And the region between the 526th to 1555th bases is deleted! / Amplified by The following oligo DNA primers were used in this PCR. [0107] SQ-GPD1-R0: CAGCTTTTTCCAAATCAGAGAGAGCAG (酉 己酉 lj number 11) mut-CLuc-CFl: TCTCTGGCCTCTGTGGAGATCTTAAAATGA (SEQ ID NO: 12) The composition of this PCR reaction solution was as follows: KOD plus DNA polymerase 1 μ 1; 10x KOD plus buffer 5 ^ 1; 2 mM each dNTP mixture 5 ^ 1; 25 mM magnesium sulfate 2 μ ΐ; SQ-GPD1-R0 (SEQ ID NO: lXlOpmol / l) 1.5 1; mut-CLuc-CFl (SEQ ID NO: 12) (lOpmol / 1) 1.5 1; Plasmid pCLuRA_TDH3 solution (lng / ^ 1) 1 ^ 1; Sterile water 33 μ1.

 [0108] PCR was performed at 94 ° C for 2 minutes (inactivation of anti-polymerase antibody) for 1 cycle, and at 94 ° C for 15 seconds (degenerative), 50 ° C for 30 seconds (annealing), and 68 ° C. A cycle of 7 minutes (extension) was performed in 30 cycles. A portion of the PCR reaction solution obtained by this PCR reaction was electrophoresed with 1% agarose. As a result, a DNA fragment of about 6.5 kbp was confirmed. The remaining PCR reaction solution was purified with Sigma GenElute ™ PCR Clean-Up Kit, ethanol precipitated, and then dissolved in 50 1 TE buffer to obtain “DNA solution E”.

 [0109] The DNA fragment contained in each of DNA solution D and DNA solution E thus obtained has the sequence between the 460th to 525th bases in the base sequence shown in SEQ ID NO: 4 and the Share the sequence between the 1556th to 1663th bases!

 [0110] Saccharomyces cerevisiae generally undergoes homologous recombination with high probability in cells. Therefore, if DNA solution D and DNA solution E were simultaneously introduced into Saccharomyces cerevisiae, a point mutation was introduced into the region encoding Saccharomyces cerevisiae that contains the circular DNA (α1 factor-derived secretory signal peptide. The mutant plasmid pCLuRA-TDH3) is reconstituted by homologous recombination, and Saccharomyces cerevisiae can be transformed with this reconstituted plasmid.

 [0111] Thus, Saccharomyces cerevisiae BY4743 A PRB vermillion was transformed using an equal volume mixture of DNA solution D and DNA solution E. Transformation was performed using the Frozen-EZ Yeast Transformation II from Zymo Research according to the product protocol.

 [0112] 1-2. Screening of secretory signal peptide gene derived from mutant α 1 factor

Saccharomyces transformed as described in Section 1-1 above, cerevisiae with synthetic agar plate without uracil (0.67% Yeast nitrogen base without amino acids (Bettaton Dickinson), 40 μg / ml adenine, 20 μ g / ml L_arginine monohydrochloride, 100 μ g / ml L_aspara Formic acid, 100 ^ g / ml sodium L-glutamate monohydrate, 20 ^ g / ml L-histidine, 60μg / ml L-leucine, 30μg / ml L_lysine hydrochloride, 20μg / ml L_methionine, 50 μg / ml L-phenylalanine, 375 ag / ml L-serine, 200 μg / ml L-threonine, 40 μg / ml L-tryptophan, 30 ^ g / ml L- Tyrosine, 150 ^ g / ml L-valine, 30 ^ g / ml L-isoleucine, 2% D-glucose and 2% agar: Hereinafter, simply applied to “SD-ura agar medium” and 30 ° After culturing at C for 3 days, colonies of transformants (hereinafter simply referred to as “mutants”) were formed.

 [0113] Using a colony picker device, each colony of the mutant was added to a synthetic liquid medium (SD-ura agar medium with agar removed and a final concentration of 200 mM potassium phosphate buffer (pH 6.0) added: Then, simply called “buffered SD-ura medium”) was inoculated into a 96-well deep well plate (2 ml volume of each well) in which 0.95 ml was dispensed to each well. However, 6 of the 96 wells were mutated as controls! /, Na! /, Saccharomyces cerevisiae BY4743 Δ PRB vermilion transformed with plasmid pCLuRA-TDH3 (hereinafter simply “wild type”) Was called).

 [0114] Deep well plates inoculated with mutant and wild-type Saccharomyces cerevisiae were cultured at 30 ° C for 48 hours with shaking at 1200rpm. Thereafter, the culture medium was transplanted in a 96-well format as it was to a 96-well deep well plate (2 ml volume of each well) in which 0.95 ml of the same medium was dispensed to each well at 50 to 1 per well. Next, the deep wall plate containing the transplanted culture solution was subjected to shaking culture at 1200 rpm for 48 hours at 30 ° C.

 [0115] After culturing, the deep well plate was centrifuged, and the culture supernatant for each well was transferred to a 96-well plate (black) as it was in a 96-well format.

 [0116] Next, using Bertoold Centro LB 960, each well was subjected to 80 1 calories of umi firefly luciferin solution (Ι Μ ミ firefly luciferin, lOOmM Tris-HCl, ρΗ7 · 5), and emission intensity (accumulated for 1 second) ) Was measured.

[0117] An example of the results is shown in FIG. FIG. 1 shows the relative luminescence intensity (RLU) for luciferase (CLuc) in each mutant and wild type culture supernatant. Each bar is the result of one mutant or wild type. The six bars at the right end show the relative emission intensity of the wild type, and the others show the relative emission intensity of the mutants! [0118] Among the mutants as shown in Fig. 1, mutants having a relative luminescence intensity that was at least 3 times the average value of the relative luminescence intensity exhibited by the wild type 6 clones were selected.

 [0119] 1-3. Identification of amino acid substitutions in mutants with a relative luminescence intensity more than 3 times that of the wild type From the mutants selected in Section 1-2 above, Takara Bio's “Gen Torukun Yeast” was used. The DNA containing the plasmid was extracted and purified.

 [0120] Next, the extracted and purified DNA was used to transform E. coli DH5 strain. The transformed E. coli was smeared on LB (10 g / l NaCl, lOg / 1 Bac to Trypton, 5 g / l yeast extract) agar plate medium containing ampicillin sodium (100 g / ml) to form colonies.

 [0121] A plasmid was extracted from each colony thus formed by a conventional method and purified, and the nucleotide sequence between the 1st to 2700th bases in SEQ ID NO: 4 was examined. As a result, amino acid substitutions of the following mutants were identified (in the following description, for example, `` G79A '' means that the 79th glycine (G) in the amino acid sequence shown in SEQ ID NO: 2 is alanine (A)). (The one-letter code of amino acids is a symbol determined by the International Pure Chemical Association of Applied Chemistry (IUPAC)).

 [0122] (1) A19V mutant (hereinafter referred to as “pCLuRA-TDH3 [a A19 ¥]”).

 (2) P21L / D43V / E45G mutant

 (3) V22F mutant (hereinafter referred to as “pCLuRA-TDH3 [a V22 F]”)

 (4) T24A mutant (hereinafter referred to as “pCLuRA-TDH3 [a T24 8]”).

 (5) A20T / I33V mutant

 (6) V22A / T26T mutant (Here, “T26T” means that there is a base sequence encoding threonine at position 26! /, With no amino acid substitution! /, There is a base substitution)

 (7) N23Y / L64W mutant

Regarding the plasmids pCLuRA-TDH3 [a A19V], pCLuRA-TDH3 [a V22F] and pCLuRA-TDH3 [a T24A], as a result of examining the entire nucleotide sequence, mutations other than the base substitution corresponding to the indicated amino acid substitution were found. It was confirmed! [0123] 1-4. Construction of plasmid pCLuRA- TDH3 [a P21L]

 A P21L single amino acid substitution mutant plasmid (hereinafter referred to as “pCL uRA-TDH3 [a P21L]”) was prepared as follows.

[0124] First, a linear DNA fragment of pCLuRA-TDH3 [aP21L] was prepared by PCR. The following oligo DNA primers were used for PCR.

[0125] P21L-for: CTAGTCAACACTACAACAGAAGATG (SEQ ID NO: 13)

 P21-rev: AGCAGCTAATGCGGAGGATGCT (SEQ ID NO: 14)

 The sequence “CTA” at the 5 ′ end of primer P21L-for is a sequence that replaces the proline codon with a leucine codon.

 [0126] The composition of the PCR reaction solution was as follows: KOD plus DNA polymerase (Toyobo) 0.4 μ 1; 10x KOD plus buffer 2 μ \; 2 mM each dNTP mixture 2 μ \; 25 mM magnesium sulfate 0.8 1; P21L-for (SEQ ID NO: 1 SXlOpmol / 1) 0.6 〃 1; P21_rev (SEQ ID NO: 1 4) (10 pmol / 1) 0.6 1; Plasmid pCLuRA_TDH3 solution (lng / 1) 1 μ 1; Sterile water 12.6 1

Yes

 [0127] PCR was performed at 94 ° C for 2 minutes (inactivation of anti-polymerase antibody) for one cycle, and at 94 ° C for 15 seconds (denaturation), at 50 ° C for 30 seconds (annealing), and at 68 ° C. A cycle of 8 minutes (extension) was performed in 30 cycles. The entire PCR reaction solution obtained by this PCR was electrophoresed with 1% agarose. As a result, a DNA fragment of about 7.5 kbp was confirmed. This DNA fragment was purified by Sigma GenElute ™ MINUS EtBr SPIN COLUMNS and ethanol precipitation.

 [0128] Next, both 5 'ends of the obtained DNA fragment were phosphorylated with T4 polynucleotide kinase. This was ligated with T4 DNA ligase as a DNA substrate and circularized. The circularized DNA was ethanol precipitated and then dissolved in 50 1 TE buffer.

 [0129] Furthermore, PCR was performed again using this circularized DNA as a cage. The primers used were as follows.

 [0130] FAR-f: AACCCTCACTAAAGGGAACAAAAGCTGGCT (IJ Number 15)

mut-CLuc-R: AACTCCTTCCTTTTCGGTTAGAGCGGATGT (SEQ ID NO: 16) The composition of this PCR reaction solution was as follows: KOD plus DNA polymerase (Toyobo) 1 μ 1; 10x KOD plus buffer 5 ^ 1; 2 mM each dNTP mixture 5 ^ 1; 25 mM sulfate Gnesium 2 1; FAR-f (SEQ ID NO: ΧΐΟ δρρπιοΙ / ^ Ι) 1.5 1; mut-CLuc-R (SEQ ID NO: 1 Q) (l0pmo \ / μ \) 1.5 μ \; the above circular DNA solution 1 1; sterilization Water 33 1.

[0131] PCR was performed at 94 ° C for 2 minutes (inactivation of anti-polymerase antibody) for 1 cycle, and at 94 ° C for 15 seconds (degenerative), 50 ° C for 30 seconds (annealing), and 68 ° C. A cycle of 3 minutes (extension) was performed in 30 cycles.

 [0132] The DNA fragment obtained by this PCR is a region between the first to the 2663th base in the base sequence shown in SEQ ID NO: 4.

 [0133] Next, a portion of the PCR reaction solution obtained by this PCR was electrophoresed with 1% agarose. As a result, a DNA fragment of about 2.5 kbp was confirmed. The remaining PCR reaction solution was purified by Sigma Gen Elute ™ PCR Clean-Up Kit and ethanol precipitation.

 [0134] The purified DNA fragment was ligated with restriction enzymes BamHI (recognition site starting with the 40th base in the base sequence shown in SEQ ID NO: 4) and Xbal (2576th in the base sequence shown in SEQ ID NO: 4). After double digestion with 6 bases starting from the base, the whole amount was electrophoresed with 1% agarose. After electrophoresis, a DNA fragment of about 2.5 kbp was purified by Sigma GenElute ™ MINUS EtBr SPIN COLUMNS and ethanol precipitation, and dissolved in TE buffer to give “DNA lyophilization night F”.

 On the other hand, the plasmid pCLuRA-TDH3 was similarly double digested with restriction enzymes BamHI and Xbal, and then electrophoresed with 1% agarose. After electrophoresis, a DNA fragment of about 5 kbp was purified by Sigma GenElute ™ MINUS EtBr SPIN COLUMNS and ethanol precipitation and dissolved in TE buffer solution to obtain “DNA solution G”.

 [0136] Next! /, DNA solution F and DNA solution G were mixed in equal amounts and ligated with T4 DNA ligase.

 This reaction solution was used to transform E. coli DH5a strain. The transformed E. coli was smeared on an LB agar plate medium containing ampicillin sodium (100 g / ml) to form colonies.

 [0137] As a result of extracting and purifying the plasmid from the formed colony by a conventional method and examining the entire base sequence, the base substitution corresponding to the intended P21L substitution occurred, and other mutations occurred. Confirmed not.

[0138] In this way, pCLuRA-TDH3 [a P21L] was prepared. [0139] 1-5. Construction of plasmid pCLuRA- TDH3 [a A20T]

 A20T single amino acid substitution mutant plasmid (hereinafter referred to as “pCL uRA-TDH3 [aA20T]”) was prepared as follows.

 [0140] First, a linear DNA fragment of pCLuRA-TDH3 [aA20T] was prepared by PCR. The following oligo DNA primers were used for PCR.

 [0141] A20T— c: ACTCCAGTCAACACTACAACAGA (IJ No. 17)

 A20-rev: AGCTAATGCGGAGGATGCTGCG (SEQ ID NO: 18)

 The sequence “ACT” at the 5 ′ end of primer A20T-C is a sequence that replaces the alanine codon with a threonine codon.

 [0142] The composition of this PCR reaction solution was as follows: KOD plus DNA polymerase (Toyobo) 0.4 μ 1; 10x KOD plus buffer 2 μ \; 2 mM each dNTP mixture 2 μ \; 25 mM magnesium sulfate 0.8 1; A20T-C (SEQ ID NO: 17) (10 0101/1) 0.6 1; A20_rev (SEQ ID NO: 18) (lOpmol / 1) 0.6 1; Plasmid pCLuRA_TDH3 solution (lng / 1) 1 μ 1; Sterile water 12.6 1 .

 [0143] PCR was performed at 94 ° C for 2 minutes (inactivation of anti-polymerase antibody) for one cycle, and at 94 ° C for 15 seconds (degenerative), at 48 ° C for 30 seconds (annealing), and at 68 ° C. A cycle of 8 minutes (extension) was performed in 30 cycles. The entire PCR reaction solution obtained by this PCR was electrophoresed with 1% agarose. As a result, a DNA fragment of about 7.5 kbp was confirmed. This DNA fragment was purified by Sigma GenElute ™ MINUS EtBr SPIN COLUMNS and ethanol precipitation.

 [0144] Next, both 5 'ends of the obtained DNA fragment were phosphorylated with T4 polynucleotide kinase. This was ligated with T4 DNA ligase as a DNA substrate and circularized. Using this reaction solution, E. coli DH5a strain was transformed. The transformed E. coli was smeared on LB agar plate medium containing ampicillin sodium (100 g / ml) to form colonies.

 [0145] As a result of extracting and purifying the plasmid from the formed colony by a conventional method and examining the entire base sequence, the base substitution corresponding to the target A20T substitution occurred, and other mutations occurred. Confirmed not.

 [0146] In this way, pCLuRA-TDH3 [aA20T] was prepared.

 [0147] 1-6. Construction of plasmids pCLuRA_TDH3 [a V22A] and pCLuRA_TDH3 [a N23Y]

In accordance with the method for preparing the plasmid pCLuRA-TDH3 [aA20T] described in Section 1-5 above, German amino acid substitution mutant plasmid (hereinafter referred to as “pCLuRA-TDH3 [a V22A]”) and N23Y single amino acid substitution mutant plasmid (hereinafter referred to as “pCLuRA-TD CL3 [α Η23Υ]”) Was made. However, the primers used in PCR were pCLuRA-ΤDH3 [a V22A]!

[0148] V22A: GCTAACACTACAACAGAAGATGAAA (SEQ ID NO: 19)

 V22-rev- c: TGGAGCAGCTAATGCGGAGGAT (SEQ ID NO: 20)

 On the other hand, the primers used in PCR for pCLuRA-TDH3 [aN23Y] were as follows.

 [0149] N23Y: TACACTACAACAGAAGATGAAACGG (SEQ ID NO: 21)

 N23- rev- c: GACTGGAGCAGCTAATGCGGAG (SEQ ID NO: 22)

 As a result of examining the entire nucleotide sequence of each plasmid, it was confirmed that a base substitution corresponding to the target amino acid substitution occurred! /, And that other mutations occurred! /, No! /

Yes

 [0150] In this way, pCLuRA-TDH3 [a V22A] and pCLuRA-TDH3 [a N23Y] were obtained.

[0151] 1-7. Reproduction Experiment (1)

 Plasmids pCLuRA—TDH3, pCLuRA— TDH3 [a A19V], pCLuRA—TDH3 [α P21L], p

Saccharomyces cerevisiae BY4743 Δ PRB vermilion was transformed with CLuRA-TDH3 [a V22F] and pCLuRA-TDH3 [a T24A], respectively.

[0152] After transformation, arbitrarily select three colonies on SD-ura agar medium.

Each well (well) of a 6-well deep well plate (well 2 ml volume) was inoculated into 0.95 ml of buffered SD-ura medium. 30 deep well plates after inoculation

The culture was shaken at 1200 rpm for 48 hours at ° C.

[0153] After culturing, the culture medium was transplanted in a 96-well format as it was to a 96-well deep well plate (2 ml of each well) in which 0.91 ml of the same medium was dispensed to each well.

 [0154] Next, the deep well plate containing the transplanted culture solution was subjected to shaking culture at 1200 rpm for 30 hours at 30 ° C.

[0155] After incubation, transfer 100 〃 1 each from each well culture solution to a transparent 96-well plate. Absorbance (ie, cell density) was measured (the blank is buffered SD-ura medium). Further, the deep well plate was centrifuged, and the relative luminescence intensity (RLU) of luciferase (CLuc) in the culture supernatant was measured by the same method as described in Section 1-2 above. The results are shown in Table 1 below.

 [Table 1] Luminescence values

 Plasmid variant OD600 Luminescence value (relative value)

 (Relative value) / OD600

0.714 0.635 1.12 pCLuRA-TDH3 Wild type 0.596 0.558 1.01

 0.551 0.639 0.86

 9.678 0.635 15.24 pCLuRA-TDH3 [aA19V] A19V 4.884 0.632 7.73

 6.424 0.636 10.1

 5.097 0.616 8.27 pCLuRA-TDH3 [aP21 L] P21 L 4.741 0.624 7.6

 6.544 0.663 9.87

 4.447 0.615 7.23 pCLuRA-TDH3 [aV22F] V22F 3.011 0.634 4.75

 2.831 0.653 4.34

 2.35 0.619 3.8 pCLuRA-TDH3 [aT24A] T24A 2.369 0.627 3.78

 3.096 0.642 4.82

[0157] As is clear from Table 1, compared to the wild type (plasmid pCLuRA-TDH3), A19V mutant (plasmid pCLuRA-TDH3 [a A19V]) and P21L mutant (plasmid pCLuRA_TDH3 [a P 21L]) Per cell (OD600) at least 7 times, at least 4 times for V22F mutant (plasmid pCLuRA-TDH3 [a V22F]) and at least 3.5 times for T24A mutant (plasmid pCLuRA-TDH3 [a T24A]) An increase in luminescence value, that is, an increase in luciferase protein secretion was observed.

 [0158] 1-8. Reproduction experiment (2)

Plasmids pCLuRA-TDH3, pCLuRA-TDH3 [a A19V], pCLuRA-TDH3 [a A20T], pCLuRA_TDH3 [a P21L, pCLuRA_TDH3 [a V22A, pCLuRA_TDH3 [a V22F, pCL uRA-TDH3 [H N23Y] [aT24A] was used to transform Saccharomyces cerevisiae BY4743 Δ PRB vermillion, respectively. [0159] After transformation, colonies on the SD-ura agar medium were scraped and inoculated into each well of a deep well plate to which 1 ml of buffered SD-ura medium had been dispensed in advance. Each mutant (transformant) was inoculated with 6 wells. The deep well plate after inoculation was cultured with shaking at 1200 rpm for 45 hours at 30 ° C.

 [0160] After culturing, the culture solution was transplanted in a 96-well format as it was to a 96-well deep well plate in which 0.91 ml of the same medium was dispensed to each well.

 [0161] Next, the deep well plate containing the transplanted culture solution was subjected to shaking culture at 1200 rpm for 30 hours at 30 ° C.

 [0162] After culturing, 50 1 from each culture solution of each well was transferred to a transparent 96-well plate, and the absorbance at 600 nm (ie, cell density) was measured. Further, the deep well plate was centrifuged, and the relative luminescence intensity (RLU) of luciferase (C Luc) in the culture supernatant was measured by the same method as described in Section 1-2 above. However, the concentration of luciferin to be dispensed was 5. In addition, the relative luminescence intensity was divided by the cell density and normalized so that the wild-type value would be 1.

 [0163] The results are shown in Figure 2 and Table 2 below. In FIG. 2, each bar represents the result of each mutant or wild type shown, and the horizontal axis represents the luminescence value per OD (OD600). The results in Fig. 2 and Table 2 are the average values and standard deviations from the respective cultures derived from 6 wells (N = 6) transplanted after each mutant or wild type was inoculated in the above. is there.

 [Table 2] Luminescence values

 Plasmid luminescence value (relative value)

 Variant OD600 standard deviation

(Relative value) / OD600 (N = 6) pCLuRA-TDH3 wild type 0.321 0.321 1.00 0.11 pCLuRA-TDH3 [aA19V] A19V 5.709 0.293 19.52 0.52 pCLuRA-TDH3 [aA20T] A20T 2.024 0.296 6.85 0.26 pCLuRA-TDH3 [aP21 L] P21 L 5.827 0.290 20.12 1.72 pCLuRA-TDH3 [aV22F] V22F 2.445 0.291 8.40 0.75 pCLuRA-TDH3 [aV22A] V22A 4.431 0.304 14.58 1.43 pCLuRA-TDH3 [aN23Y] N23Y 1.528 0.272 5.63 0.58 pCLuRA-TDH3 0.24 0.24 0.24 2 and Table 2 reveal that the A1 9V mutant (plasmid pCLuRA-TDH3 [a A19V]) and the P21L mutant (plasmid pCLuRA-TD H3 [a P21L) compared to the wild type (plasmid pCLuRA-TDH3) ]) About 20 times, V22A mutant (plasmid pCLuRA-TDH3 [a V22A]) about 1 5-fold, A20T mutant (plasmid pCLuRA-TDH3 [a A20T]), V22F mutant (plasmid pCLu RA-TDH3 [a V22F]), N23Y mutant (plasmid pCLuRA_TDH3 [a N23Y]) and T24A mutant (plasmid pCLuRA) -TDH3 [aT24A]) also showed an increase in luminescence value per microbial mass (OD600) of 5 times or more, that is, an increase in luciferase protein secretion. All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.

Claims

The scope of the claims  [1] The following secretion signal peptide (a) or (b):  (a) In the amino acid sequence described in SEQ ID NO: 2, the 19th alanine, the 20th alanine, the 21st proline, the 22nd valine, the 23rd asparagine and the 24th threonine Secretory signal peptide consisting of an amino acid sequence in which at least one amino acid selected from the group consisting of is replaced with another amino acid  (b) In the amino acid sequence of the secretion signal peptide of (a) above, an amino acid sequence in which one or several amino acids are deleted, substituted or added at positions other than the 19th to 24th amino acids. A secretory signal peptide that improves the secretory production of a protein linked to the C-terminal side by 3 times or more compared to a secretory signal peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2  [2] The 19th alanine is replaced with another amino acid! /, The secretion signal peptide according to 1. [3] The 19th alanine force S-parin is substituted, 2. The secretion signal peptide according to 2. [4] The 20th alanine is replaced with another amino acid! /, The secretion signal peptide according to 1. [5] The secretory signal peptide according to claim 1 or 4, wherein the 20th alanine is substituted with threonine. [6] The 21st proline is substituted with another amino acid, The secretion signal peptide according to 1. [7] The secretory signal peptide according to claim 1 or 6, wherein the 21st proline is substituted with leucine. [8] The secretion signal peptide according to claim 1, wherein the 22nd palin is replaced with another amino acid! /. [9] The secretion signal peptide according to claim 1 or 8, wherein the 22nd valine is substituted with phenylalanin or alanine! /. [10] The above-mentioned 23rd asparagine is substituted with another amino acid. The secretion signal peptide according to claim 1. [11] The secretory signal peptide according to claim 1 or 10, wherein the 23rd asparagine is substituted with tyrosine. [12] The secretion signal peptide according to claim 1, wherein the 24th threonine is substituted with another amino acid. [13] The secretory signal peptide according to claim 1 or 12, wherein the 24th threonine is substituted with alanine. [14] DNA encoding the secretory signal peptide according to any one of claims 1 to 13; [15] A DNA fragment comprising the DNA of claim 14. [16] A recombinant vector comprising the DNA of claim 14.  [17] A fusion protein obtained by linking the secretory signal peptide according to any one of claims;! To 13 and a foreign protein.  [18] The fusion protein according to claim 17, wherein the foreign protein is linked to the C-terminal side of the secretory signal peptide! /. [19] DNA encoding the fusion protein according to claim 17 or 18. [20] A DNA fragment comprising the DNA of claim 19. [21] A recombinant vector comprising the DNA of claim 19.  [22] A transformant comprising the DNA fragment according to claim 20 or the recombinant vector according to claim 21.  [23] The transformant according to claim 22, wherein the host is yeast. [24] The transformant according to claim 23, wherein the yeast is Saccharomyces cerevisiae.  [25] A protein production method, wherein the transformant according to any one of claims 22 to 24 is cultured, and the foreign protein is secreted or expressed in a membrane.