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WO2008032659A1 - Peptide signal de sécrétion doté d'une efficacité améliorée, et procédé de production de protéine au moyen dudit peptide - Google Patents

Peptide signal de sécrétion doté d'une efficacité améliorée, et procédé de production de protéine au moyen dudit peptide Download PDF

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Publication number
WO2008032659A1
WO2008032659A1 PCT/JP2007/067525 JP2007067525W WO2008032659A1 WO 2008032659 A1 WO2008032659 A1 WO 2008032659A1 JP 2007067525 W JP2007067525 W JP 2007067525W WO 2008032659 A1 WO2008032659 A1 WO 2008032659A1
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Prior art keywords
signal peptide
amino acid
dna
protein
secretory signal
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Japanese (ja)
Inventor
Kosei Kawasaki
Satoru Ohgiya
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National Institute of Advanced Industrial Science and Technology AIST
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National Institute of Advanced Industrial Science and Technology AIST
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to a secretory signal peptide and a protein production method using them.
  • 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.
  • 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]”! /).
  • secretory signal peptide the sequence is called “secretory signal coordination 1]”! /.
  • 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)
  • 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.
  • 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: [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.
  • the target protein 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.
  • 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.
  • the secretory expression system is suitable for mass production of proteins and is often used practically for the production of pharmaceuticals.
  • the N-terminus of the target protein can be obtained as a natural protein.
  • 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.
  • ⁇ 1 factor ( ⁇ 1 mating factor) of budding yeast Saccharomyces cerevisiae is a secreted protein and has a secretory signal peptide.
  • 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.
  • a prosequence consisting of a sequence.
  • the pre-sequence and the pro-sequence are collectively referred to as pre-pro-binding IJ.
  • a sequence containing a force prepro sequence whose pre sequence is a secretory signal sequence is often referred to as a secretory signal sequence.
  • the synthesized nascent ⁇ 1 factor has a secretory signal peptide containing the above-mentioned preb mouth sequence on the heel end side.
  • the ⁇ 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 ⁇ ⁇ 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.
  • 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.
  • mature ⁇ 1 factor will be synthesized.
  • the synthesized mature ⁇ 1 factor is secreted extracellularly.
  • secretory signal peptides derived from Saccharomyces cerevisiae proteins include secretory signal peptides derived from proteins such as ⁇ -factor receptor, invertase, and phosphatase, in addition to the above-mentioned secretory signal peptides derived from ⁇ 1 factor.
  • 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.
  • the ⁇ 1 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.
  • 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- ⁇ 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).
  • secretory signal peptide derived from ⁇ factor is also used in an expression system (Invitrogen) using methanol yeast Pichia pastoris.
  • the secretory signal peptide derived from ⁇ 1 factor can be widely applied to secrete and express a protein.
  • Patent Document 1 discloses an invention relating to modification of a pre-sequence.
  • 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.
  • leucine which is a hydrophobic amino acid.
  • 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.
  • Patent Document 2 discloses an invention relating to modification of a prosequence.
  • glycosylation site 2 includes one glycosylation site (eg, the 23rd asparagine in SEQ ID NO: 2) of the three pro sequences derived from ⁇ 1 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.
  • IJ glycosylation recognition sequence
  • Patent Document 1 Japanese Patent No. 2564536
  • Patent Document 2 Japanese Patent No. 2793215
  • Patents l Emr, 3 ⁇ 4.D., Schekman, R., rlessel, ⁇ ., 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, ⁇ _ ⁇ ⁇ , “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, ⁇ ⁇ 135- 151
  • Non-Patent Document 6 Xiong, AS, Yao, QH, Peng, RH, Han, P ⁇ ., Cheng, ZM, Li, Y., jOURNAL OF APPLIED MICROBIOLOGYJ, 2005, Vol. 98, No.
  • 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.
  • 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 ⁇ 1 factor of Saccharomyces cerevisiae Peptide power
  • 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 ⁇ 1 factor, and the present invention was completed. It came to do.
  • the present invention includes the following.
  • the secretion signal peptide according to 1) or (10).
  • (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.
  • 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.
  • FIG. 1 shows luciferase (CLuc) in each mutant and wild-type culture supernatant.
  • V shows the relative emission intensity
  • FIG. 2 shows luciferase (CLuc) in each mutant and wild-type culture supernatant.
  • V shows the luminescence value (relative value) per microbial mass (OD600).
  • the secretory signal peptide according to the present invention is a variant of the secretory signal peptide derived from ⁇ 1 factor of Saccharomyces cerevisiae. Specifically, it is the following secretory signal peptide (a) or (b).
  • the peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 is a secretory signal peptide derived from Saccharomyces cerevisiae ⁇ 1 factor.
  • 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 ⁇ 1 factor.
  • 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.
  • 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 ⁇ 1 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).
  • a host such as yeast compared to the secretory signal peptide derived from wild type ⁇ 1 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.).
  • substitution at the amino acid position described above may be single or a combination of two or more.
  • the other amino acids are! / Other than the specific amino acids of the secretion signal peptide derived from the wild-type ⁇ 1 factor at each position described above, and may be misplaced amino acids! /.
  • Ni! / The following amino acids are preferably substituted.
  • 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 ⁇ 1 factor
  • positions other than the 19th to 24th amino acids include the 18th and 25th amino acids.
  • 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 ⁇ 1 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.
  • the secretory signal peptide according to the present invention can be made into a fusion protein linked to a foreign protein.
  • the foreign protein means a protein exogenous to the secretory signal peptide according to the present invention.
  • the peptide is also ⁇ 3 ⁇ 4.
  • the foreign protein is not particularly limited, and may be any naturally occurring secreted protein, membrane protein, or other non-secretory protein.
  • 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.
  • 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.
  • 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.
  • 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.
  • foreign proteins examples include various hormones (such as insulin) of proteins or peptides, erythropoietin, cytodynamic force (such as interferon), and various enzymes (such as invertase and amylase).
  • hormones such as insulin
  • erythropoietin erythropoietin
  • cytodynamic force such as interferon
  • enzymes such as invertase and amylase
  • 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.
  • 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").
  • DNA encoding the fusion protein according to the present invention DNA encoding the fusion protein according to the present invention.
  • the foreign protein in the fusion protein can be secreted or expressed on a membrane.
  • 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
  • DNA encoding the fusion protein in preparation of DNA encoding the fusion protein according to the present invention, the analysis according to the present invention is performed.
  • a DNA encoding a secretory signal peptide 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 ⁇ 1 factor-derived secretory signal peptide
  • the wild type ⁇ 1-factor-derived secretory signal peptide coding region is amplified by PCR using.
  • 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.
  • a known mutagenesis method for example, Kunkel method or PCR using a synthetic oligonucleotide as a primer for mutagenesis
  • DNA encoding the secretory signal peptide according to the present invention can be chemically synthesized.
  • 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.
  • 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.
  • 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.
  • an enzyme DNA ligase
  • double-stranded DNA having sequences already linked may be chemically synthesized.
  • 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.
  • a shuttle vector for example, a DNA distribution that allows the vector to replicate autonomously in Escherichia coli. ⁇ 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.
  • 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).
  • 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.
  • examples of such a promoter include a promoter derived from a TDH3 (glyceroanolaldehyde triphosphate dehydrogenase) gene.
  • 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.
  • 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.
  • 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.
  • DNA encoding a foreign protein to be secreted or membrane expressed is appropriately inserted into the DNA fragment or recombinant vector.
  • 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.
  • 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.
  • Saccharomyces cerevisiae ⁇ 1 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 j3 ⁇ 4, the genus Mucor and the like.
  • 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.
  • electroporation electroporation (elect mouth position method), spheroplast method, Examples include the lithium acetate method.
  • 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.
  • 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. ! /
  • DNA having homology with the host chromosomal DNA sequence is added to both ends thereof,
  • DNA having homology with the host chromosomal DNA sequence is added to both ends thereof,
  • 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.
  • 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.
  • 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.
  • using a primer previously labeled with a fluorescent dye, etc. CR can be used to detect the amplification product.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the foreign protein is a membrane protein
  • 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.
  • 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.
  • 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.
  • 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.
  • CLuc luciferase
  • Saccharomyces cerevisiae was used as the host.
  • 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.
  • This plasmid pCLuRA-TDH3 is a secreted signal peptide derived from wild type ⁇ 1 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).
  • a CLuc gene a gene encoding a fusion protein
  • the plasmid pCLuRA_TDH3 has a saccharomyces.
  • Cerevisiae TDH3 (systematic gene name: YGR192C) gene promoter operably incorporated upstream (5 ') of the a CLuc gene.
  • YGR192C systematic gene name: YGR192C gene promoter operably incorporated upstream (5 ') of the a CLuc gene.
  • 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.
  • 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 ⁇ 1-factor-derived secretory signal peptide (corresponding to the secretory signal peptide according to the present invention)
  • sgl-f CACCAAGAACTTAGTTTCGAGGG (IJ No. 5)
  • the composition of this Error Prone PCR reaction solution was as follows: Taq DNA polymerase (Roche, 5 unit / ⁇ 1) 1 ⁇ ⁇ ; lOxPCR Duffer without magnesium ion 10 ⁇ 1; Deoxynucleotide mixed solution for Error Prone PCR 10 ⁇ 1; 25 mM magnesium chloride 28 ⁇ 1; 5 mM manganese chloride 5.0 1; Plasmid pCLuRA-TDH3 solution (15 ⁇ 3 ⁇ 4 / 1) 1 ⁇ 1; sgl-i (SEQ ID NO: 5 XlOpmol / a 1) 3 ⁇ 1; sg2_r (SEQ ID NO: 6) (10 pmol / ⁇ 1) 3 1; Sterile water 39 ⁇ 1.
  • composition of the above-mentioned error-prone PCR-use doxynucleotide mixed solution was as follows: lOOmM dCTP 100 ⁇ 1; lOOmM dTTP 100 ⁇ 1; lOOmM dGTP 20 ⁇ 1; 100 mM dATP 20 a 1; sterilized water 760 ⁇ l.
  • 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.
  • 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 ⁇ 1; 10x KOD plus buffer 2 ⁇ ⁇ ; 2 mM each dNTP mixture 2 ⁇ ⁇ ; 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 ⁇ 1; Sterile water 12.6
  • 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 TM MINUS EtBr SPIN COLUMNS and ethanol precipitation and dissolved in 10-1 TE buffer to obtain “DNA solution B”.
  • 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 ⁇ ⁇ ; 2mM each dNTP mixture 2 ⁇ ⁇ ; 25 mM magnesium sulfate 0.8 a 1; sg2-f (SEQ ID NO: 9) (10 pmol / ⁇ 1) 0.6 ⁇ 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 ⁇ 1.
  • 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.
  • a DNA fragment of about 700 bp was confirmed. This DNA fragment was purified by Sigma GenElute TM MINUS EtBr SPIN COLUMNS and ethanol precipitation, and dissolved in 10-1 TE buffer to obtain “DNA solute night C”.
  • 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.
  • 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 ⁇ 1 factor-derived secretory signal peptide. Contains DNA molecules.
  • composition of this PCR reaction solution was as follows: KOD plus DNA polymerase 1 ⁇ 1;
  • 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.
  • a cycle of 1 minute 20 seconds (extension) was performed in 30 cycles.
  • the remaining PCR reaction solution was purified by Sigma GenElute TM PCR Clean-Up Kit and ethanol precipitation, dissolved in 50 1 TE buffer, and used as “DNA Purification Night D”.
  • SQ-GPD1-R0 CAGCTTTTTCCAAATCAGAGAGCAG ( ⁇ ⁇ 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 ⁇ 1; 10x KOD plus buffer 5 ⁇ 1; 2 mM each dNTP mixture 5 ⁇ 1; 25 mM magnesium sulfate 2 ⁇ ⁇ ; 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 ⁇ 1.
  • 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 TM PCR Clean-Up Kit, ethanol precipitated, and then dissolved in 50 1 TE buffer to obtain “DNA solution E”.
  • 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!
  • 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 ( ⁇ 1 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.
  • 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.
  • 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.
  • 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”
  • 6 of the 96 wells were mutated as controls! /, Na! /, Saccharomyces cerevisiae BY4743 ⁇ PRB vermilion transformed with plasmid pCLuRA-TDH3 (hereinafter simply “wild type”) was called).
  • 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.
  • 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.
  • 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.
  • RLU relative luminescence intensity
  • 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.
  • 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.
  • 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)).
  • G 79th glycine
  • A alanine
  • A19V mutant (hereinafter referred to as “pCLuRA-TDH3 [a A19 ⁇ ]”).
  • V22F mutant (hereinafter referred to as “pCLuRA-TDH3 [a V22 F]”)
  • T24A mutant (hereinafter referred to as “pCLuRA-TDH3 [a T24 8]”).
  • 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)
  • a P21L single amino acid substitution mutant plasmid (hereinafter referred to as “pCL uRA-TDH3 [a P21L]”) was prepared as follows.
  • a linear DNA fragment of pCLuRA-TDH3 [aP21L] was prepared by PCR.
  • the following oligo DNA primers were used for PCR.
  • sequence “CTA” at the 5 ′ end of primer P21L-for is a sequence that replaces the proline codon with a leucine codon.
  • the composition of the PCR reaction solution was as follows: KOD plus DNA polymerase (Toyobo) 0.4 ⁇ 1; 10x KOD plus buffer 2 ⁇ ⁇ ; 2 mM each dNTP mixture 2 ⁇ ⁇ ; 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 ⁇ 1; Sterile water 12.6 1
  • 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 TM MINUS EtBr SPIN COLUMNS and ethanol precipitation.
  • 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.
  • 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 ⁇ 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: ⁇ ⁇ / ⁇ ⁇ ) 1.5 1; mut-CLuc-R (SEQ ID NO: 1 Q) (l0pmo ⁇ / ⁇ ⁇ ) 1.5 ⁇ ⁇ ; the above circular DNA solution 1 1; sterilization Water 33 1.
  • 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.
  • 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.
  • 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 TM MINUS EtBr SPIN COLUMNS and ethanol precipitation, and dissolved in TE buffer to give “DNA lyophilization night F”.
  • 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 TM MINUS EtBr SPIN COLUMNS and ethanol precipitation and dissolved in TE buffer solution to obtain “DNA solution G”.
  • 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.
  • A20T single amino acid substitution mutant plasmid (hereinafter referred to as “pCL uRA-TDH3 [aA20T]”) was prepared as follows.
  • a linear DNA fragment of pCLuRA-TDH3 [aA20T] was prepared by PCR.
  • the following oligo DNA primers were used for PCR.
  • A20T— c ACTCCAGTCAACACTACAACAGA (IJ No. 17)
  • sequence “ACT” at the 5 ′ end of primer A20T-C is a sequence that replaces the alanine codon with a threonine codon.
  • composition of this PCR reaction solution was as follows: KOD plus DNA polymerase (Toyobo) 0.4 ⁇ 1; 10x KOD plus buffer 2 ⁇ ⁇ ; 2 mM each dNTP mixture 2 ⁇ ⁇ ; 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 ⁇ 1; Sterile water 12.6 1 .
  • 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 TM MINUS EtBr SPIN COLUMNS and ethanol precipitation.
  • 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.
  • German amino acid substitution mutant plasmid hereinafter referred to as “pCLuRA-TDH3 [a V22A]”
  • pCLuRA-TD CL3 [ ⁇ ⁇ 23 ⁇ ] German amino acid substitution mutant plasmid
  • pCLuRA-TD CL3 [ ⁇ ⁇ 23 ⁇ ] N23Y single amino acid substitution mutant plasmid
  • V22A GCTAACACTACAACAGAAGATGAAA (SEQ ID NO: 19)
  • V22-rev- c TGGAGCAGCTAATGCGGAGGAT (SEQ ID NO: 20)
  • primers used in PCR for pCLuRA-TDH3 [aN23Y] were as follows.
  • N23Y TACACTACAACAGAAGATGAAACGG (SEQ ID NO: 21)
  • N23- rev- c GACTGGAGCAGCTAATGCGGAG (SEQ ID NO: 22)
  • Saccharomyces cerevisiae BY4743 ⁇ PRB vermilion was transformed with CLuRA-TDH3 [a V22F] and pCLuRA-TDH3 [a T24A], respectively.
  • the culture was shaken at 1200 rpm for 48 hours at ° C.
  • 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.
  • the deep well plate containing the transplanted culture solution was subjected to shaking culture at 1200 rpm for 30 hours at 30 ° C.
  • 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 ⁇ PRB vermillion, respectively.
  • 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.
  • the deep well plate containing the transplanted culture solution was subjected to shaking culture at 1200 rpm for 30 hours at 30 ° C.
  • each bar represents the result of each mutant or wild type shown, and the horizontal axis represents the luminescence value per OD (OD600).

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Abstract

La présente invention concerne un peptide signal de sécrétion doté d'une haute efficacité de sécrétion. Ledit peptide signal renferme une séquence d'acides aminés comportant la substitution d'un résidu d'acide aminé spécifique par un autre résidu d'acide aminé présent dans la séquence d'acides aminés d'un peptide signal de sécrétion dérivé du facteur α1 de Saccharomyces cerevisiae.
PCT/JP2007/067525 2006-09-14 2007-09-07 Peptide signal de sécrétion doté d'une efficacité améliorée, et procédé de production de protéine au moyen dudit peptide Ceased WO2008032659A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014101300A (ja) * 2012-11-19 2014-06-05 Chiba Univ 脳送達用キャリアおよびその用途
JP2016537993A (ja) * 2014-02-28 2016-12-08 ノヴォ ノルディスク アー／エス アルファ接合因子プロペプチドバリアント
WO2025037535A1 (fr) * 2023-08-11 2025-02-20 株式会社Ｃｏ２資源化研究所 Gène peptidique de signal de sécrétion dérivé d'une bactérie du genre hydrogenophilus
EP4304360A4 (fr) * 2021-03-11 2025-10-29 Tenza Inc Peptides signal synthétiques pour diriger la sécrétion de protéines hétérologues dans la levure

Citations (1)

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JPH022339A (ja) * 1987-12-30 1990-01-08 Chiron Corp 先端を切断したα―因子リーダー配列を用いた酵母における異種タンパクの改良された発現および分泌

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JPH022339A (ja) * 1987-12-30 1990-01-08 Chiron Corp 先端を切断したα―因子リーダー配列を用いた酵母における異種タンパクの改良された発現および分泌

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LI H. ET AL.: "Impact of Amino Acid Changes in the Signal Peptide on the Secretion of the Tat-dependent Xylanase C from Streptomyces lividans", FEMS MICROBIOL. LETT., vol. 255, no. 2, February 2006 (2006-02-01), pages 268 - 274, XP003019983 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014101300A (ja) * 2012-11-19 2014-06-05 Chiba Univ 脳送達用キャリアおよびその用途
JP2016537993A (ja) * 2014-02-28 2016-12-08 ノヴォ ノルディスク アー／エス アルファ接合因子プロペプチドバリアント
US12024542B2 (en) 2014-02-28 2024-07-02 Novo Nordisk A/S Mating factor alpha pro-peptide variants
EP4304360A4 (fr) * 2021-03-11 2025-10-29 Tenza Inc Peptides signal synthétiques pour diriger la sécrétion de protéines hétérologues dans la levure
WO2025037535A1 (fr) * 2023-08-11 2025-02-20 株式会社Ｃｏ２資源化研究所 Gène peptidique de signal de sécrétion dérivé d'une bactérie du genre hydrogenophilus

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