JPWO2008032659A1 - Efficient secretion signal peptides and protein production methods using them - Google Patents

Abstract

The present invention relates to a secretory signal peptide having a high secretion efficiency, comprising an amino acid sequence in which a specific amino acid is substituted with another amino acid in the secretory signal peptide derived from Saccharomyces cerevisiae α1 factor.

Description

  The present invention relates to secretory signal peptides and protein production methods using them.

  Some proteins are synthesized in the cell and then remain inside the cell, while others are released outside the cell. Proteins released outside the cell are generally called secreted proteins. In addition, there is a protein that is synthesized in a cell and then penetrates into a cell membrane, an endoplasmic reticulum membrane or the like, or exists in a partially inserted form. These proteins are called membrane proteins.

  It is known that secretory 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 sequence is called “secretory signal peptide” (the sequence is called “secretory signal sequence”). The importance of secretory signal peptides in the production of secreted proteins (extracellular secretion) and membrane proteins (membrane localization) is by removing secretory signal sequences from the amino acid sequences of secreted proteins and membrane proteins. They are widely recognized because they no longer show extracellular secretion or membrane localization.

  In addition, when the secretory signal peptide of a secreted protein or 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 the membrane) can be observed. It is known that secreted proteins or membrane proteins substituted with secretory 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.

  The secretory production of a protein using a secretory signal peptide has the following advantages.

  (1) In an intracellular expression system for proteins produced and accumulated in cells, the accumulated target protein is often degraded by proteolytic enzymes present in the cells. On the other hand, in a protein secretory expression system using a secretory signal peptide, the target protein is secreted into the culture medium, so that there is relatively little risk of degradation or the like.

  (2) In a protein intracellular expression system, cells must be disrupted in order to purify the target protein, and all substances in the cell become contaminants. On the other hand, in the protein secretory expression system using the 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, ie, the culture solution. The target protein is relatively easy to purify.

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

  (4) In a protein secretory expression system using a secretory signal peptide, the N-terminus of the target protein can be obtained as a natural protein. On the other hand, in the intracellular expression system in which the protein stays in the cell, the N-terminus is often methionine or formylmethionine, which is not always the same as the natural protein, and the biochemical activity is also low. It may be different from the natural type.

  The α1 factor (α1 mating factor) of the budding yeast Saccharomyces cerevisiae is a secreted protein and has a secretory signal peptide. Specifically, the α1 factor-derived secretory signal peptide is a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2. The secretory signal peptide includes a pre-sequence composed of the first to 19th amino acid sequences and a pro-sequence composed of the 20th to 89th amino acid sequences in SEQ ID NO: 2. The pre-sequence and the pro sequence are collectively referred to as a pre-pro sequence. In a narrow sense, the pre-sequence is a secretory signal sequence, but a sequence containing a pre-pro sequence is often called a secretory signal sequence.

  The synthesized nascent α1 factor has a secretory signal peptide containing the above-mentioned prepro sequence on the N-terminal side. The α1 factor having a secretory signal peptide synthesized in the cell is cleaved by an endopeptidase in the process of translocation into the endoplasmic reticulum to become an α1 factor precursor. Next, the pro sequence is cleaved by endoplasmic reticulum Kex2 endopeptidase at a site represented by a peptide bond between the 85th arginine and the 86th glutamic acid of SEQ ID NO: 2. Furthermore, it was cleaved by the Golgi Ste13 endopeptidase at 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, and after these processings As a result, mature α1 factor is synthesized. The synthesized mature α1 factor is secreted extracellularly.

  As the secretion signal peptide derived from the protein of Saccharomyces cerevisiae, in addition to the secretion signal peptide derived from α1 factor described above, for example, a secretion signal peptide derived from a protein such as α factor receptor, invertase, phosphatase, and the like is known, It has been used so far. However, these secretory signal peptides have commonality in terms of their ability to secrete proteins and peptides connected to the C-terminal side (in the case of membrane proteins, they are produced on the membrane), but to what extent The efficiency (hereinafter referred to as “secretion efficiency”) of whether a quantity of protein or peptide can be transported out of the cell and secreted (in the case of membrane protein, can be produced in the membrane) is not the same. In fact, among them, the α1 factor-derived secretory signal peptide is very often used because of its high secretory efficiency, and many achievements relating to secretory production of various proteins are known.

  Examples of proteins secreted and produced using a secretory signal peptide derived from α1 factor include, for example, invertase (Non-patent Document 1), epidermal growth factor (Non-patent Document 2), interferon-α1 (Non-patent Document 3), β -Endorphins (Non-Patent Document 4), anti-CD33 single chain antibodies (Non-Patent Document 5), phytases (Non-Patent Document 6) and the like are known. Moreover, it is known that the secretion signal peptide derived from α1 factor functions as a secretion signal peptide not only in Saccharomyces cerevisiae but also in other yeast species and can secrete proteins (Non-patent Document 7). Furthermore, α1 factor-derived secretory signal peptides are also used in expression systems (Invitrogen) using the methanol yeast Pichia pastoris. Thus, the secretory signal peptide derived from α1 factor can be widely applied to secrete and express a protein.

  By the way, an attempt has been made to improve the secretion efficiency of the linked protein by modifying the pre-sequence or pro-sequence derived from the secreted protein or membrane protein.

  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 sequence (in a narrow sense) of egg white lysozyme is substituted with leucine, which is a hydrophobic amino acid. According to the description of 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 is expressed. The amount of secreted protein is increased as compared with the case of using it. However, the increase in secretion amount is only about 2.5 times.

  On the other hand, Patent Document 2 discloses an invention related to modification of a prosequence. Patent Document 2 discloses one glycosylation site (for example, the 23rd asparagine in SEQ ID NO: 2) and its glycosylation recognition sequence (the 23rd asparagine) of the three pro-sequences derived from α1 factor. And the subsequent 2 amino acids), the gene coding for the pro sequence from which part or all of the pro sequence on the C-terminal side has been deleted and the Kex2 cleavage site on the 3 'end side It was disclosed that a gene encoding a fusion protein was constructed in which a gene encoding insulin was linked and a gene encoding an insulin-like substance protein was further linked to the 3 ′ end thereof. According to the description in Patent Document 2, when a gene encoding the fusion protein is expressed in Saccharomyces cerevisiae, the amount of the protein secreted is increased as compared with the case where a natural prosequence is used. However, the increase in secretion is less than 3 times, and it is difficult to say that it has improved dramatically.

  In recent years, as a means of providing protein samples for the elucidation of biochemical phenomena as seen in the “Protein 3000” project of the Ministry of Education, Culture, Sports, Science and Technology, as well as the production of proteins as pharmaceuticals and industrial enzymes, There is a strong demand for the development of protein expression systems that can be produced easily and in large quantities. For this reason, there is a need for further improvement in the secretory expression system of proteins in terms of improving the secretion efficiency.

In addition, it is generally known that when a membrane protein is synthesized, a mechanism common to secreted proteins, such as passage through the endoplasmic reticulum membrane by a signal sequence, is used. Therefore, it is considered that the secretory signal peptide described above can be adapted to efficiently produce a membrane protein.
Japanese Patent No. 2564536 Japanese Patent No. 2793215 Emr, SD, Schekman, R., Flessel, MC, Thorner, J., `` Proceedings of the National Academy of Sciences of the United States of America '', 1983, 80, p.7080-7084 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 '', 1984, 81, p.4642-4646 Singh, A., Lugovoy, J., Kohr, W., Perry, LJ, `` Nucleic Acids Research '', 1984, Vol. 12, No. 23, p.8927-8938 Bitter, GA, Chen, KK, Banks, AR, Lai, PH., `` Proceedings of the National Academy of Sciences of the United States of America '', 1984, 81, p. 5330-5334 Emberson, LM, Trivett, AJ, Blower, PJ, Nicholls, PJ, `` JOURNAL OF IMMUNOLOGICAL METHODS '', 2005, Vol.305, No.2, p.135-151 Xiong, AS, Yao, QH, Peng, RH, Han, PL, Cheng, ZM, Li, Y., `` JOURNAL OF APPLIED MICROBIOLOGY '', 2005, Vol. 98, No. 2, p.418-428 Kalandadze, A., Galleno, M., Foncerrada, L., Strominger, JL, Wucherpfennig, KW, "THE JOURNAL OF BIOLOGICAL CHEMISTRY", 1996, 271, 33, p.20156-20162

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

  Accordingly, 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.

  As a result of earnest research to solve the above problems, a secretory signal peptide composed of an amino acid sequence in which a specific amino acid residue is substituted with another amino acid in a secretory signal peptide derived from Saccharomyces cerevisiae α1 factor is a natural type. As compared with the secretory signal peptide derived from α1 factor, the secretory production amount of the protein linked to the C-terminal side can be improved at least 3 times, and the present invention has been completed.

  The present invention includes the following.

  (1) The following secretion signal peptide (a) or (b):

(a) A group consisting of the 19th alanine, the 20th alanine, the 21st proline, the 22nd valine, the 23rd asparagine and the 24th threonine in the amino acid sequence described in SEQ ID NO: 2 A secretory signal peptide consisting of an amino acid sequence in which at least one selected amino acid is replaced with another amino acid
(b) in the amino acid sequence of the secretion signal peptide of (a) above, consisting of 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. And a secretory signal peptide that improves the secretory production amount of the protein linked to the C-terminal side by 3 times or more compared to the secretory signal peptide consisting of the amino acid sequence of SEQ ID NO: 2. (2) The 19th alanine is The secretory signal peptide according to (1), which is substituted with another amino acid.

  (3) The secretion signal peptide according to (1) or (2), wherein the 19th alanine is substituted with valine.

  (4) The secretion signal peptide according to (1), wherein the 20th alanine is substituted with another amino acid.

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

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

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

  (8) The secretory signal peptide according to (1), wherein the 22nd valine is substituted with another amino acid.

  (9) The secretory signal peptide according to (1) or (8), wherein the 22nd valine is substituted with phenylalanine or alanine.

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

  (11) The secretory signal peptide according to (1) or (10), wherein the 23rd asparagine is substituted with tyrosine.

  (12) The secretion signal peptide according to (1), wherein the 24th threonine is substituted with another amino acid.

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

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

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

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

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

  (18) The fusion protein according to (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 (17) or (18).

  (20) A DNA fragment containing the DNA according to (19).

  (21) A recombinant vector comprising the DNA according to (19).

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

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

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

  (25) A protein production method, wherein the transformant according to any one of (22) to (24) is cultured, and the foreign protein is secreted or expressed in a membrane.

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

  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.

FIG. 1 shows the relative luminescence intensity for luciferase (CLuc) in each mutant and wild type culture supernatant. FIG. 2 shows the luminescence value (relative value) per bacterial cell amount (OD600) for luciferase (CLuc) in each mutant and wild type culture supernatant.

  Hereinafter, the present invention will be described in detail.

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

(a) A group consisting of the 19th alanine, the 20th alanine, the 21st proline, the 22nd valine, the 23rd asparagine and the 24th threonine in the amino acid sequence described in SEQ ID NO: 2 A secretory signal peptide consisting of an amino acid sequence in which at least one selected amino acid is replaced with another amino acid;
(b) in the amino acid sequence of the secretion signal peptide of (a) above, consisting of 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. And a secretory signal peptide that improves the secretory production amount of the protein linked to the C-terminal side by 3 times or more as compared with a secretory signal peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2.

  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 to 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.

  The secretion signal peptide described in (a) above is the 19th alanine, the 20th alanine, the 21st proline, the 22nd in the α1 factor-derived secretion signal peptide (amino acid sequence 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 valine, the 23rd asparagine and the 24th threonine is substituted with another amino acid. By this amino acid substitution, the secretory production amount of the protein linked to the C-terminal side in the host such as yeast is compared with the secretion 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). It 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, a protein membrane linked to the C-terminal side in a host such as yeast as compared with a secretory signal peptide derived from wild-type α1 factor (that is, a peptide consisting of the amino acid sequence described in SEQ ID NO: 2) ( Localization to cell membranes, endoplasmic reticulum membranes, etc. can be improved.

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

  Here, the other amino acid may be any amino acid other than the specific amino acid of the secretory signal peptide derived from the wild-type α1 factor at each position described above, but at each position, the following amino acid is substituted. Is preferred.

(1) 19th alanine: substitution with valine, leucine or isoleucine
(2) 20th alanine: substitution with threonine or serine
(3) 21st proline: substitution with leucine, valine or isoleucine
(4) 22nd valine: substitution with phenylalanine or alanine
(5) 23rd asparagine: substitution to tyrosine or phenylalanine
(6) 24th threonine: substitution with alanine or valine

  On the other hand, the secretion signal peptide described in the above (b), in the secretion signal peptide described in (a), in the position other than the 19th to 24th amino acids, one or several (for example, 1-10) (Preferably 1 to 5, particularly preferably 1 to 3) of amino acid sequence is deleted, substituted or added, and compared to the secretory signal peptide derived from wild-type α1 factor on the C-terminal side. It is possible to improve the production of secreted protein by more than 3 times. Examples of positions other than the 19th to 24th amino acids include the 18th and 25th amino acids.

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

  The secretory signal peptide according to the present invention can be 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. The foreign protein includes a peptide.

  Here, the foreign protein is not particularly limited, and may be any secreted protein, membrane protein, or other non-secretory protein that exists in nature. However, when a non-secretory protein is linked to a secretory signal peptide, there are many cases in which no secretion is observed. Therefore, in the present invention, among non-secretory proteins, a protein that can be secreted is defined as a foreign protein. In addition, when connecting secretory protein or membrane protein, it is preferable to connect the mature protein except the secretory signal peptide which the protein has naturally to the secretory signal peptide according to the present invention. For example, the secretory signal peptide portion of the secreted protein or membrane protein can be known by comparing the deduced amino acid sequence obtained from the base sequence of cDNA by subjecting the mature protein to N-terminal amino acid analysis. Alternatively, estimation is performed by a signal peptide prediction program such as SignalP3.0 (http://www.cbs.dtu.dk/services/SignalP/ and Nielsen H. et al. (1997) Protein Engineering, 10: 1-6). Is also possible.

  Examples of foreign proteins include various hormones (such as insulin) of proteins or peptides, erythropoietin, cytokines (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.

  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.

  The DNA according to the present invention is a DNA encoding the secretory signal peptide according to the present invention or a DNA encoding the above-described fusion protein (hereinafter referred to as “DNA encoding the fusion protein according to the present invention”). In particular, by introducing a DNA fragment or a recombinant vector containing DNA encoding the fusion protein according to the present invention 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. Is possible.

  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.

  First, in preparation of DNA encoding the fusion protein according to the present invention, DNA encoding the secretory signal peptide according to the present invention and DNA encoding the foreign protein are prepared. In preparation of DNA encoding a secretory signal peptide according to the present invention, for example, PCR using primers complementary to the nucleotide sequences at both ends of the region encoding α1-factor-derived secretory signal peptide using Saccharomyces cerevisiae genomic DNA as a template To amplify the wild type α1 factor-derived secretory signal peptide coding region. Next, the amplified PCR product is subjected to a known mutagenesis method (for example, PCR using the Kunkel method or a synthetic oligonucleotide for mutagenesis as a primer) to encode the secretory signal peptide according to the present invention. Can be produced. Alternatively, it is also possible to chemically synthesize DNA encoding the secretory signal peptide according to the present invention.

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

  When the DNA encoding the secretory signal peptide according to the present invention and the DNA encoding the foreign protein are linked, they 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. The DNA encoding the foreign protein is preferably operably linked to the 3 ′ end of the DNA encoding the secretory signal peptide according to the present invention. As a linking means, an enzyme (DNA ligase) reaction is generally used. Alternatively, double-stranded DNA having sequences already linked may be chemically synthesized.

  The recombinant vector according to the present invention can be obtained by inserting the 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 replicate in the host, and examples thereof include plasmids, shuttle vectors, helper plasmids and the like. When a shuttle vector is used, for example, by including a DNA sequence that allows the vector to replicate autonomously in Escherichia coli, both the host used for protein expression and the E. coli cells And can be replicated. Further, when the vector itself does not have replication ability, it may be a DNA fragment that can be replicated by insertion into a host chromosome.

  In the recombinant vector according to the present invention, it is preferable that a promoter having transcription initiation activity in the host is operably linked to the 5 ′ end of the DNA encoding the fusion protein according to the present invention. That is, it is preferable that a promoter suitable for the host is inserted into the recombinant vector according to the present invention so as to be functionally linked to the 5 ′ end of the DNA encoding the fusion protein according to the present invention. The promoter may be inducible or non-inducible (constitutive). From the viewpoint of protein secretion productivity, a promoter whose transcription initiation activity is recognized to be strong in the host to be used, for example, a promoter derived from a gene encoding an enzyme of Saccharomyces cerevisiae glycolysis is desirable. For example, examples of such a promoter include a promoter derived from a TDH3 (glyceraldehyde triphosphate dehydrogenase) gene.

  Alternatively, the recombinant vector according to the present invention can be constructed by inserting and ligating the promoter, the DNA encoding the secretory signal peptide according to the present invention and the DNA encoding the foreign protein separately.

  On the other hand, in the DNA fragment according to the present invention, a promoter having transcription initiation activity in the host is located on the 5 ′ end side of the DNA encoding the fusion protein according to the present invention in accordance with the preparation of the recombinant vector according to the present invention described above. Preferably they are functionally linked.

  Incidentally, in accordance with the production of the above-described recombinant vector or DNA fragment according to the present invention, for example, as a foreign protein-producing DNA fragment or recombinant vector, a DNA fragment containing the DNA encoding the secretory signal peptide according to the present invention or Recombinant vectors 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.

  Further, a transformant by introducing a DNA fragment or a recombinant vector according to the present invention containing a DNA encoding the fusion protein according to the present invention (hereinafter referred to as “recombinant vector according to the present invention”) into a host. Is made. Although it does not specifically limit as a host, For example, the true fungi and animal cell containing various yeast are mentioned. The yeast may be any yeast, for example, Saccharomyces cerevisiae, Shizosaccharomyces pombe, Pichia pastoris, Candida albicans, Hansenula polymorpha. Saccharomyces cerevisiae is particularly preferable from the viewpoint that transformation methods and genetic manipulation techniques are established. In addition, it has been confirmed that the α1-factor-derived secretory signal peptide of Saccharomyces cerevisiae can function in heterologous yeasts other than Saccharomyces cerevisiae (Non-patent Document 7). Examples of other fungi include the genus Aspergillus, the genus Penicillium, the genus Trichoderma, the genus Rhizopus, and the genus Mucor.

  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 (electroporation), spheroplast method, lithium acetate method Etc. Alternatively, a YIp-based vector or a transformation method such as yeast for substitution / insertion into a chromosome may be used. Furthermore, the method of introducing the recombinant vector according to the present invention into eubacteria or animal cells containing yeast or the like may be any method described in general academic books or academic papers.

  In addition, 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, a DNA having homology with the host chromosomal DNA sequence is added to both ends thereof, without using a vector. In addition, it can be incorporated into the chromosomal DNA of the host cell by the homologous recombination function inherent in 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.

  Whether or not the recombinant vector according to the present invention has been incorporated into the host can be confirmed by PCR, Southern hybridization, Northern hybridization or the like. For example, DNA is prepared from the transformant, PCR is performed by designing a DNA-specific primer. Thereafter, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide, SYBR (registered trademark) Green solution, etc., and the amplified product is detected as a band. Confirm that it has been transformed. Moreover, PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product. Furthermore, 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.

  Next, the obtained transformant is cultured under conditions that allow it to grow. In any case, it is desirable that the culture conditions for the transformant are set in consideration of the characteristics of the host and the stability of the desired foreign protein. For example, as shown in this example, when the budding firefly luciferase (corresponding to a foreign protein) is secreted and expressed using the budding yeast Saccharomyces cerevisiae as a host, the culture is performed so that the yeast grows and the luciferase is not inactivated. The 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.

  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. For example, the culture solution is subjected to centrifugation. The culture supernatant thus obtained can be measured using the enzyme activity, physiological activity, etc. of the foreign protein as indicators. In addition, the amount of foreign protein secreted can be measured by a general immunological technique such as Western blotting or ELISA using an antibody specific to the target foreign protein. Alternatively, a method using a fluorescence microscope or flow cytometry may be used.

  On the other hand, when the foreign protein is a membrane protein, the transformant is separated, and then the membrane fraction is separated, so that it is applied to the membrane such as a cell membrane according to the above-described method for measuring the secretory protein production. The amount of localization 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.

  In this way, the desired foreign protein secreted is produced from the culture supernatant by conventional protein purification methods such as ammonium sulfate salting out, ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, high performance liquid chromatography. , Affinity chromatography, isoelectric focusing, polyacrylamide gel electrophoresis, and the like.

  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 secreted protein expression system has the advantage that the initial crude sample, that is, the culture solution contains fewer contaminants other than the desired protein and is relatively easy to purify, compared to the expression system that accumulates the desired protein in the cell. .

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.

  In this example, as a foreign protein secreted by the improved secretory function of the secretory signal peptide according to the present invention, a luciferase (hereinafter referred to as `` CLuc '') derived from Cypridina noctiluca is used. It was.

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

  The expression plasmid pCLuRA-TDH3 is an expression vector for secreting and expressing CLuc in Saccharomyces cerevisiae.International Publication No. 2006/132350 (International Application PCT / JP2006 / 311597; Japanese Patent Application No. 2005-169768 takes precedence. On the basis of rights).

  This plasmid pCLuRA-TDH3 is composed of a secreted signal peptide derived from a wild type α1 factor (amino acid sequence: SEQ ID NO: 2) and a mature protein of CLuc (amino acid sequence 1-18 in CLuc amino acid sequence shown in SEQ ID NO: 3) And a gene encoding a fusion protein (hereinafter referred to as “αCLuc gene”).

  Furthermore, in the plasmid pCLuRA-TDH3, the promoter of the Saccharomyces cerevisiae TDH3 (systematic gene name: YGR192C) gene is operably incorporated upstream (5 ′ side) of the αCLuc gene. Therefore, by operating this promoter, the αCLuc gene is expressed, and CLuc is secreted outside the cell by the function of the α1 factor-derived secretory signal peptide.

  The plasmid pCLuRA-TDH3 contains a DNA sequence necessary for plasmid replication that functions in E. coli and an ampicillin resistance gene. Thus, this plasmid is a shuttle vector that is replicated and maintained in both Saccharomyces cerevisiae and E. coli.

  The base sequence shown in SEQ ID NO: 4 is a partial base sequence of the plasmid pCLuRA-TDH3, and shows the promoter of the TDH3 gene, the αCLuc gene, and the 5 ′ side and 3 ′ side 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, a region encoding the α1 factor-derived secretory signal peptide (from the 701st to the 955th in the nucleotide sequence shown in SEQ ID NO: 4) Error Prone PCR (error-prone PCR) was performed using a region corresponding to the base) as a target. In this Error Prone PCR, the following oligo DNA primers were used.

sg1-f: CACCAAGAACTTAGTTTCGAGGG (SEQ ID NO: 5)
sg2-r: TCAGGTTCGTAAGGACAGTCCTG (SEQ ID NO: 6)
The composition of this Error Prone PCR reaction solution was as follows: Taq DNA polymerase (Roche, 5 unit / μl) 1 μl; 10 × PCR buffer without magnesium ion 10 μl; Deoxynucleotide mixed solution 10 μl for Error Prone PCR; 25 mM chloride Magnesium 28 μl; 5 mM manganese chloride 5.0 μl; Plasmid pCLuRA-TDH3 solution (150 ng / μl) 1 μl; sg1-f (SEQ ID NO: 5) (10 pmol / μl) 3 μl; sg2-r (SEQ ID NO: 6) (10 pmol / μl) 3 μl ; 39 μl of sterile water.

  The composition of the above-mentioned Error Prone PCR deoxynucleotide mixed solution was as follows: 100 mM dCTP 100 μl; 100 mM dTTP 100 μl; 100 mM dGTP 20 μl; 100 mM dATP 20 μl;

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

Subsequently, the entire PCR reaction solution obtained by Error Prone PCR was electrophoresed with 1% agarose. As a result, a DNA fragment of about 300 bp was confirmed. This DNA fragment was purified by Sigma GenElute ^™ MINUS EtBr SPIN COLUMNS and ethanol precipitation and dissolved in 10 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to obtain “DNA solution A”. “DNA solution A” contains DNA molecules into which random point mutations have been introduced by Error Prone PCR.

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

SQ-GPD1-F0: CATGTATCTATCTCATTTTCTTAC (SEQ ID NO: 7)
sg1-r: CCCTCGAAACTAAGTTCTTGGTG (SEQ ID NO: 8)
The composition of this PCR reaction solution was as follows: KOD plus DNA polymerase (Toyobo) 0.4 μl; 10 × KOD plus buffer 2 μl; 2 mM each dNTP mixture 2 μl; 25 mM magnesium sulfate 0.8 μl; SQ-GPD1-F0 ( SEQ ID NO: 7) (10 pmol / μl) 0.6 μl; sg1-r (SEQ ID NO: 8) (10 pmol / μl) 0.6 μl; Plasmid pCLuRA-TDH3 solution (1 ng / μl) 1 μl; Sterile water 12.6 μl.

PCR consists of one cycle of 2 minutes at 94 ° C (inactivation of anti-polymerase antibody) and 15 seconds at 94 ° C (denaturation), 30 seconds at 50 ° C (annealing) and 1 minute at 68 ° C (extension). Performed in 30 cycles. As a result of electrophoresis of the entire PCR reaction solution obtained by this PCR using 1% agarose, a DNA fragment of about 250 bp was confirmed. This DNA fragment was purified by Sigma GenElute ^™ MINUS EtBr SPIN COLUMNS and ethanol precipitation and dissolved in 10 μl of TE buffer to obtain “DNA solution B”.

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

sg2-f: CAGGACTGTCCTTACGAACCTGA (SEQ ID NO: 9)
SQ-CLuc-CR1: TGGACAACCGTCAAACTCCTGGTTGATCTT (SEQ ID NO: 10)
The composition of this PCR reaction solution was as follows: KOD plus DNA polymerase 0.4 μl; 10 × KOD plus buffer 2 μl; 2 mM each dNTP mixture 2 μl; 25 mM magnesium sulfate 0.8 μl; sg2-f (SEQ ID NO: 9) (10 pmol) 0.6 μl; SQ-CLuc-CR1 (SEQ ID NO: 10) (10 pmol / μl) 0.6 μl; Plasmid pCLuRA-TDH3 solution (1 ng / μl) 1 μl; Sterile water 12.6 μl.

PCR consists of one cycle of 2 minutes at 94 ° C (inactivation of anti-polymerase antibody) and 15 seconds at 94 ° C (denaturation), 30 seconds at 50 ° C (annealing) and 1 minute at 68 ° C (extension). 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 μl of TE buffer to obtain “DNA solution C”.

  Next, PCR was performed using the obtained mixture of DNA solution A, DNA solution B and DNA solution C as a template. The DNA fragment obtained by this PCR corresponds to the region between the 460th to 1663th bases in the base sequence shown in SEQ ID NO: 4. However, since DNA solution A obtained by Error Prone PCR is used as a part of the template, the region encoding the α1-factor-derived secretory signal peptide contains a DNA molecule into which a point mutation has been introduced.

  The composition of this PCR reaction solution was as follows: KOD plus DNA polymerase 1 μl; 10 × KOD plus buffer 5 μl; 2 mM each dNTP mixture 5 μl; 25 mM magnesium sulfate 2 μl; SQ-GPD1-F0 (SEQ ID NO: 7) (10 pmol) 1.5 μl; SQ-CLuc-CR1 (SEQ ID NO: 10) (10 pmol / μl) 1.5 μl; DNA solution A 3 μl; DNA solution B 1 μl; DNA solution C 1 μl;

PCR consists of 1 minute at 94 ° C for 2 minutes (inactivation of anti-polymerase antibody) and 15 seconds at 94 ° C (denaturation), 30 seconds at 50 ° C (annealing) and 1 minute 20 seconds at 68 ° C (extension). The cycle 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 and dissolved in 50 μl of TE buffer to obtain “DNA solution D”.

  On the other hand, in the base sequence of plasmid pCLuRA-TDH3, a region lacking the region between the 526th to 1555th bases in the base sequence shown in SEQ ID NO: 4 was amplified by PCR. The following oligo DNA primers were used in this PCR.

SQ-GPD1-R0: CAGCTTTTTCCAAATCAGAGAGAGCAG (SEQ ID NO: 11)
mut-CLuc-CF1: TCTCTGGCCTCTGTGGAGATCTTAAAATGA (SEQ ID NO: 12)
The composition of the PCR reaction solution was as follows: KOD plus DNA polymerase 1 μl; 10 × KOD plus buffer 5 μl; 2 mM each dNTP mixture 5 μl; 25 mM magnesium sulfate 2 μl; SQ-GPD1-R0 (SEQ ID NO: 11) (10 pmol) 1.5 μl; mut-CLuc-CF1 (SEQ ID NO: 12) (10 pmol / μl) 1.5 μl; plasmid pCLuRA-TDH3 solution (1 ng / μl) 1 μl; sterile water 33 μl.

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

  The DNA fragments contained in each of DNA solution D and DNA solution E thus obtained have a sequence between the 460th to 525th bases in the base sequence shown in SEQ ID NO: 4 and the 1556th to 5th bases. The sequence between the 1663th base is shared.

  Saccharomyces cerevisiae generally undergoes homologous recombination with high probability in cells. Therefore, if DNA solution D and DNA solution E are simultaneously introduced into Saccharomyces cerevisiae, a circular plasmid (a mutant plasmid pCLuRA in which a point mutation has been introduced into the region encoding the secretory signal peptide derived from the α1 factor in Saccharomyces cerevisiae) -TDH3) is reconstituted by homologous recombination, and Saccharomyces cerevisiae can be transformed with this reconstituted plasmid.

  Therefore, Saccharomyces cerevisiae BY4743ΔPRB1 strain was transformed using an equal volume mixture of DNA solution D and DNA solution E. Transformation was performed using Frozen-EZ Yeast Transformation II from Zymo Research according to the product protocol.

1-2. Screening for Secretory Signal Peptide Gene Derived from Mutant α1-Factor Saccharomyces cerevisiae transformed in Section 1-1 above was synthesized from a synthetic agar plate (0.67% Yeast nitrogen base without amino acids) containing no uracil. Dickinson), 40 μg / ml adenine, 20 μg / ml L-arginine monohydrochloride, 100 μg / ml L-aspartic acid, 100 μg / ml L-glutamic acid sodium monohydrate, 20 μg / ml L-histidine, 60 μg / ml L- Leucine, 30 μg / ml L-lysine hydrochloride, 20 μg / ml L-methionine, 50 μg / ml L-phenylalanine, 375 μg / ml L-serine, 200 μg / ml L-threonine, 40 μg / ml L-tryptophan, 30 μg / ml L -Tyrosine, 150 μg / ml L-valine, 30 μg / ml L-isoleucine, 2% D-glucose and 2% agar: Hereinafter, simply applied to “SD-ura agar medium” and cultured at 30 ° C. for 3 days Transformants (hereinafter simply referred to as “mutants”) Colonies) were formed.

  For each colony of the mutant, using a colony picker device, a synthetic liquid medium (agar is removed from SD-ura agar medium and a final concentration of 200 mM potassium phosphate buffer (pH 6.0) is added: A 96-well deep well plate (2 ml in each well) in which 0.95 ml of “buffered SD-ura medium” was dispensed in each well was inoculated. However, 6 of the 96 wells were inoculated with a Saccharomyces cerevisiae BY4743ΔPRB1 strain (hereinafter simply referred to as “wild type”) transformed with a plasmid pCLuRA-TDH3 that was not mutated as a control.

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

  After the culture, the deep well plate was centrifuged, and 20 μl of the culture supernatant per well was transferred to the 96-well plate (black) as it was in the 96-well format.

  Next, 80 μl of Cypridina luciferin solution (1 μM Cypridina luciferin, 100 mM Tris-HCl, pH 7.5) was added to each well using Bertoled Centro LB 960, and the luminescence intensity (1 second integration) was measured.

  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.

  From the mutants as shown in FIG. 1, mutants having a relative luminescence intensity of 3 times or more of the average relative luminescence intensity exhibited by the wild type 6 clones were selected.

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 Sections 1-2 above, using Takara Bio's “Gen Torukun Yeast” plasmids The contained DNA was extracted and purified.

  Subsequently, Escherichia coli DH5α strain was transformed using the extracted and purified DNA. The transformed E. coli was smeared on LB (10 g / l NaCl, 10 g / l Bacto Trypton, 5 g / l yeast extract) agar plate medium containing ampicillin sodium (100 μg / ml) to form colonies.

  A plasmid was extracted and purified from each formed colony by a conventional method, 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 specified (in the following description, for example, “G79A” means that the 79th glycine (G) is substituted with alanine (A) in the amino acid sequence shown in SEQ ID NO: 2. (The one-letter symbols for amino acids are symbols defined by the International Union of Pure and Applied Chemistry (IUPAC)).

(1) A19V mutant (hereinafter, the plasmid obtained from this mutant is referred to as “pCLuRA-TDH3 [αA19V]”)
(2) P21L / D43V / E45G mutant
(3) V22F mutant (hereinafter, the plasmid obtained from this mutant is referred to as “pCLuRA-TDH3 [αV22F]”)
(4) T24A mutant (hereinafter, the plasmid obtained from this mutant is referred to as “pCLuRA-TDH3 [αT24A]”)
(5) A20T / I33V mutant
(6) V22A / T26T mutant (where `` T26T '' means that there is a base substitution without amino acid substitution in the base sequence encoding threonine at position 26)
(7) N23Y / L64W mutant In addition, as a result of examining the entire nucleotide sequence of plasmids pCLuRA-TDH3 [αA19V], pCLuRA-TDH3 [αV22F] and pCLuRA-TDH3 [αT24A], the base substitution corresponding to the indicated amino acid substitution It was confirmed that no other mutations occurred.

1-4. Construction of Plasmid pCLuRA-TDH3 [αP21L] A P21L single amino acid substitution mutant plasmid (hereinafter referred to as “pCLuRA-TDH3 [αP21L]”) was constructed as follows.

  First, a linear pCLuRA-TDH3 [αP21L] DNA fragment was prepared by PCR. The oligo DNA primers used for PCR in the preparation were as follows.

P21L-for: CTAGTCAACACTACAACAGAAGATG (SEQ ID NO: 13)
P21-rev: AGCAGCTAATGCGGAGGATGCT (SEQ ID NO: 14)
The sequence “CTA” at the 5 ′ end of the primer P21L-for is a sequence in which the proline codon is replaced with a leucine codon.

  The composition of this PCR reaction solution was as follows: KOD plus DNA polymerase (Toyobo) 0.4 μl; 10 × KOD plus buffer 2 μl; 2 mM each dNTP mixture 2 μl; 25 mM magnesium sulfate 0.8 μl; P21L-for (SEQ ID NO: 13) (10 pmol / μl) 0.6 μl; P21-rev (SEQ ID NO: 14) (10 pmol / μl) 0.6 μl; Plasmid pCLuRA-TDH3 solution (1 ng / μl) 1 μl; Sterile water 12.6 μl.

PCR consists of one cycle of 2 minutes at 94 ° C (inactivation of anti-polymerase antibody) and 15 seconds at 94 ° C (denaturation), 30 seconds at 50 ° C (annealing) and 8 minutes at 68 ° C (extension). Performed in 30 cycles. As a result of electrophoresis of the entire PCR reaction solution obtained by this PCR using 1% agarose, 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.

  Subsequently, both 5 ′ ends of the obtained DNA fragment were phosphorylated by T4 polynucleotide kinase. This was ligated with T4 DNA ligase as a DNA substrate and circularized. The circularized DNA was subjected to ethanol precipitation and then dissolved in 50 μl of TE buffer.

  Furthermore, PCR was performed again using the circularized DNA as a template. The primers used were as follows.

FAR-f: AACCCTCACTAAAGGGAACAAAAGCTGGCT (SEQ ID NO: 15)
mut-CLuc-R: AACTCCTTCCTTTTCGGTTAGAGCGGATGT (SEQ ID NO: 16)
The composition of the PCR reaction solution was as follows: KOD plus DNA polymerase (Toyobo) 1 μl; 10 × KOD plus buffer 5 μl; 2 mM each dNTP mixture 5 μl; 25 mM magnesium sulfate 2 μl; FAR-f (SEQ ID NO: 15) (10 pmol / μl) 1.5 μl; mut-CLuc-R (SEQ ID NO: 16) (10 pmol / μl) 1.5 μl; 1 μl of the above circularized DNA solution; 33 μl of sterilized water.

  PCR consists of 1 cycle at 94 ° C for 2 minutes (inactivation of anti-polymerase antibody) and 15 seconds at 94 ° C (denaturation), 30 seconds at 50 ° C (annealing) and 3 minutes at 68 ° C (extension). Performed in 30 cycles.

  The DNA fragment obtained by this PCR is a region between the 1st to 2663th bases in the base sequence shown in SEQ ID NO: 4.

Next, as a result of electrophoresis of a part of the PCR reaction solution obtained by this PCR using 1% agarose, a DNA fragment of about 2.5 kbp was confirmed. The remaining PCR reaction solution was purified by Sigma GenElute ^™ PCR Clean-Up Kit and ethanol precipitation.

The purified DNA fragment begins with restriction enzymes BamHI (recognition site starting from the 40th base in the base sequence shown in SEQ ID NO: 4) and XbaI (2576th base in the base sequence shown in SEQ ID NO: 4) After double digestion with 6 bases (recognition site), 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 obtain “DNA solution F”.

On the other hand, plasmid pCLuRA-TDH3 was similarly double digested with restriction enzymes BamHI and XbaI, 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 to obtain “DNA solution G”.

  Next, equal amounts of DNA solution F and DNA solution G were mixed and ligated with T4 DNA ligase. Escherichia coli DH5α strain was transformed with this reaction solution. The transformed E. coli was smeared on a LB agar plate medium containing ampicillin sodium (100 μg / ml) to form colonies.

  As a result of extracting and purifying the plasmid from the formed colony by a conventional method and examining the entire base sequence, it was confirmed that the base substitution corresponding to the target P21L substitution had occurred and that no other mutation had occurred. did.

  In this way, pCLuRA-TDH3 [αP21L] was produced.

1-5. Construction of Plasmid pCLuRA-TDH3 [αA20T] A20T single amino acid substitution mutant plasmid (hereinafter referred to as “pCLuRA-TDH3 [αA20T]”) was constructed as follows.

  First, a linear pCLuRA-TDH3 [αA20T] DNA fragment was prepared by PCR. The oligo DNA primers used for PCR in the preparation were as follows.

A20T-c: ACTCCAGTCAACACTACAACAGA (SEQ ID NO: 17)
A20-rev: AGCTAATGCGGAGGATGCTGCG (SEQ ID NO: 18)
The sequence “ACT” at the 5 ′ end of the primer A20T-c is a sequence in which an alanine codon is replaced with a threonine codon.

  The composition of this PCR reaction solution was as follows: KOD plus DNA polymerase (Toyobo) 0.4 μl; 10 × KOD plus buffer 2 μl; 2 mM each dNTP mixture 2 μl; 25 mM magnesium sulfate 0.8 μl; A20T-c (SEQ ID NO: 17) (10 pmol / μl) 0.6 μl; A20-rev (SEQ ID NO: 18) (10 pmol / μl) 0.6 μl; plasmid pCLuRA-TDH3 solution (1 ng / μl) 1 μl; sterile water 12.6 μl.

PCR consists of one cycle of 2 minutes at 94 ° C (inactivation of anti-polymerase antibody) and 15 seconds at 94 ° C (denaturation), 30 seconds at 48 ° C (annealing) and 8 minutes at 68 ° C (extension). Performed in 30 cycles. As a result of electrophoresis of the entire PCR reaction solution obtained by this PCR using 1% agarose, 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.

  Subsequently, both 5 ′ ends of the obtained DNA fragment were phosphorylated by T4 polynucleotide kinase. This was ligated with T4 DNA ligase as a DNA substrate and circularized. Escherichia coli DH5α strain was transformed with this reaction solution. The transformed Escherichia coli was smeared on an LB agar plate medium containing ampicillin sodium (100 μg / ml) to form colonies.

  As a result of extracting and purifying the plasmid from the formed colony by a conventional method and examining the entire base sequence, it was confirmed that the base substitution corresponding to the target A20T substitution had occurred and that no other mutation had occurred. did.

  In this way, pCLuRA-TDH3 [αA20T] was produced.

1-6. Preparation of Plasmids pCLuRA-TDH3 [αV22A] and pCLuRA-TDH3 [αN23Y] According to the method for preparing the plasmid pCLuRA-TDH3 [αA20T] described in Section 1-5 above, the V22A single amino acid substitution mutant plasmid (hereinafter referred to as the plasmid pCLuRA-TDH3 [αN23Y]) This plasmid was referred to as “pCLuRA-TDH3 [αV22A]”) and N23Y single amino acid substitution mutant plasmid (hereinafter this plasmid was referred to as “pCLuRA-TDH3 [αN23Y]”). However, the primers used in PCR were as follows for pCLuRA-TDH3 [αV22A].

V22A: GCTAACACTACAACAGAAGATGAAA (SEQ ID NO: 19)
V22-rev-c: TGGAGCAGCTAATGCGGAGGAT (SEQ ID NO: 20)
On the other hand, the primers used in the PCR for pCLuRA-TDH3 [αN23Y] were as follows.

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 intended amino acid substitution had occurred and that no other mutation had occurred.

  In this way, pCLuRA-TDH3 [αV22A] and pCLuRA-TDH3 [αN23Y] were obtained.

1-7. Reproduction Experiment (1)
Saccharomyces cerevisiae BY4743ΔPRB1 strains were transformed with plasmids pCLuRA-TDH3, pCLuRA-TDH3 [αA19V], pCLuRA-TDH3 [αP21L], pCLuRA-TDH3 [αV22F] and pCLuRA-TDH3 [αT24A], respectively.

  After transformation, 3 colonies on the SD-ura agar medium were arbitrarily selected, and 0.95 ml was dispensed into each well of a 96-well deep well plate (2 ml volume for each well). -Inoculated each in ura medium. The deep well plate after inoculation was shake-cultured at 1200 rpm for 48 hours at 30 ° C.

  After culturing, the culture solution was transplanted as it was in a 96-well format into a 96-well deep well plate (2 ml volume of each well) in which 0.95 ml of the same medium was dispensed into each well.

  Subsequently, the deep well plate containing the transplanted culture solution was subjected to shaking culture at 1200 rpm for 48 hours at 30 ° C.

  After the culture, 100 μl of the culture solution in each well was transferred to a transparent 96-well plate, and the absorbance at 600 nm (that is, the cell density) was measured (the blank is buffered SD-ura medium). Furthermore, 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.

  As is clear from Table 1, the A19V mutant (plasmid pCLuRA-TDH3 [αA19V]) and the P21L mutant (plasmid pCLuRA-TDH3 [αP21L]) are at least 7-fold compared to the wild type (plasmid pCLuRA-TDH3). , Enhancement of luminescence value per cell (OD600) at least 4 times in the V22F mutant (plasmid pCLuRA-TDH3 [αV22F]) and at least 3.5 times in the T24A mutant (plasmid pCLuRA-TDH3 [αT24A]), ie, luciferase An increase in protein secretion was observed.

1-8. Reproduction Experiment (2)
Plasmids pCLuRA-TDH3, pCLuRA-TDH3 [αA19V], pCLuRA-TDH3 [αA20T], pCLuRA-TDH3 [αP21L], pCLuRA-TDH3 [αV22A], pCLuRA-TDH3 [αV22F], pCLuRA-TDH3Y [αT24A] was used to transform Saccharomyces cerevisiae BY4743ΔPRB1 strain, respectively.

  After transformation, colonies on the SD-ura agar medium were scraped and inoculated into each well of a deep well plate into which 1 ml of buffered SD-ura medium had been dispensed in advance. For each mutant (transformant), 6 wells were inoculated. The deep well plate after inoculation was cultured with shaking at 1200 rpm for 45 hours at 30 ° C.

  After culturing, the culture solution was transplanted in a 96-well format as it was to a 96-well deep well plate in which 0.95 ml of the same medium was dispensed into each well, 50 μl per well.

  Subsequently, the deep well plate containing the transplanted culture solution was subjected to shaking culture at 1200 rpm for 23 hours at 30 ° C.

  After culturing, 50 μl of the culture solution in each well was transferred to a transparent 96-well plate, and the absorbance at 600 nm (ie, cell density) was measured. Furthermore, 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. However, the concentration of luciferin to be dispensed was 5 μM. In addition, the relative luminescence intensity was divided by the cell density and further normalized so that the wild-type value was 1.

The results are shown in FIG. 2 and Table 2 below. In FIG. 2, each bar represents the result of each mutant or wild type, and the horizontal axis represents the luminescence value (relative value) per bacterial cell amount (OD600). The results in FIG. 2 and Table 2 are the average value and standard deviation from each culture solution derived from 6 wells (N = 6) transplanted after each mutant or wild type was inoculated in the above. .

  As is clear from FIG. 2 and Table 2, the A19V mutant (plasmid pCLuRA-TDH3 [αA19V]) and the P21L mutant (plasmid pCLuRA-TDH3 [αP21L]) are compared with the wild type (plasmid pCLuRA-TDH3). About 20 times, V22A mutant (plasmid pCLuRA-TDH3 [αV22A]) about 15 times, A20T mutant (plasmid pCLuRA-TDH3 [αA20T]), V22F mutant (plasmid pCLuRA-TDH3 [αV22F]), N23Y mutant (Plasmid pCLuRA-TDH3 [αN23Y]) and T24A mutant (plasmid pCLuRA-TDH3 [αT24A]) are observed to increase luminescence value per microbial mass (OD600) more than 5 times, that is, increase luciferase protein secretion It was done.

  All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (25)

The following secretion signal peptide of (a) or (b).
(a) A group consisting of the 19th alanine, the 20th alanine, the 21st proline, the 22nd valine, the 23rd asparagine and the 24th threonine in the amino acid sequence described in SEQ ID NO: 2 A secretory signal peptide consisting of an amino acid sequence in which at least one selected amino acid is replaced with another amino acid
(b) in the amino acid sequence of the secretion signal peptide of (a) above, consisting of 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. And a secretory signal peptide that improves the secretory production amount of the protein linked to the C-terminal side more than 3 times compared to the secretory signal peptide consisting of the amino acid sequence of SEQ ID NO: 2.   The secretion signal peptide according to claim 1, wherein the 19th alanine is substituted with another amino acid.   The secretory signal peptide according to claim 1 or 2, wherein the 19th alanine is substituted with valine.   The secretion signal peptide according to claim 1, wherein the 20th alanine is substituted with another amino acid.   The secretion signal peptide according to claim 1 or 4, wherein the 20th alanine is substituted with threonine.   The secretory signal peptide according to claim 1, wherein the 21st proline is substituted with another amino acid.   The secretory signal peptide according to claim 1 or 6, wherein the 21st proline is substituted with leucine.   The secretion signal peptide according to claim 1, wherein the 22nd valine is substituted with another amino acid.   The secretion signal peptide according to claim 1 or 8, wherein the 22nd valine is substituted with phenylalanine or alanine.   The secretory signal peptide according to claim 1, wherein the 23rd asparagine is substituted with another amino acid.   The secretory signal peptide according to claim 1 or 10, wherein the 23rd asparagine is substituted with tyrosine.   The secretion signal peptide according to claim 1, wherein the 24th threonine is substituted with another amino acid.   The secretory signal peptide according to claim 1 or 12, wherein the 24th threonine is substituted with alanine.   A DNA encoding the secretory signal peptide according to any one of claims 1 to 13.   A DNA fragment comprising the DNA according to claim 14.   A recombinant vector comprising the DNA according to claim 14.   A fusion protein in which the secretory signal peptide according to any one of claims 1 to 13 and a foreign protein are linked.   The fusion protein according to claim 17, wherein the foreign protein is linked to the C-terminal side of the secretory signal peptide.   A DNA encoding the fusion protein according to claim 17 or 18.   A DNA fragment comprising the DNA according to claim 19.   A recombinant vector comprising the DNA according to claim 19.   A transformant comprising the DNA fragment according to claim 20 or the recombinant vector according to claim 21.   The transformant according to claim 22, wherein the host is yeast.   The transformant according to claim 23, wherein the yeast is Saccharomyces cerevisiae.   A method for producing a protein, 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.

Images