WO2016017736A1 - 分泌シグナルペプチドならびにそれを利用したタンパク質の分泌および細胞表層提示 - Google Patents
分泌シグナルペプチドならびにそれを利用したタンパク質の分泌および細胞表層提示 Download PDFInfo
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- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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Definitions
- the present invention relates to a secretory signal peptide and protein secretion and cell surface display using the same.
- yeasts with saccharification ability have been produced by presenting enzymes such as cellulase and hemicellulase on the cell surface.
- the cell surface presentation technique include a method using a GPI anchor protein of a cell surface localized protein.
- an expression cassette including a promoter, a secretory signal peptide, a gene for surface display, and a gene for GPI anchor protein is used.
- Patent Documents 1 to 4 There are various reports on GPI anchor proteins and promoters (Patent Documents 1 to 4 and Non-Patent Documents 1 to 5).
- a secretory signal peptide is a peptide that is localized at the cell membrane, cell wall, or present at the N-terminus of a protein secreted extracellularly.
- protein secretion expression systems it has been reported that the expression efficiency and success rate of expression can be increased by replacing the secretion signal peptide of the target protein with a highly efficient secretion signal peptide. Therefore, in the protein cell surface display system and the secretion production system, the efficiency of the secretory signal peptide is an important factor.
- Non-patent Document 6 a secretion signal peptide derived from Saccharomyces cerevisiae used in a highly efficient secretory protein expression system in existing yeast
- MF ⁇ prepro a secretion signal peptide derived from ⁇ -factor
- Patent Document 5 a secretory signal peptide derived from Saccharomyces cerevisiae, which is said to have a secretory capacity exceeding the secretory signal peptide derived from ⁇ -factor
- the secretory ability of the secretory signal peptide varies greatly depending on the combination of promoter and protein connected before and after that.
- the secretory ability of the secretory signal peptide is evaluated using only the activity when combined with a single promoter (HSP1 promoter) and a single protein (luciferase) as an index. It is.
- the secretory ability of the secretory signal peptide which is considered highly efficient in Patent Document 5, is evaluated using only the activity when the target protein is secreted extracellularly (culture supernatant) as an index.
- An object of the present invention is to provide a secretory signal peptide having a secretory capacity exceeding that of conventionally used secretory signal peptides in secretory production of proteins and cell surface display.
- the present invention provides an expression vector comprising the following (i), (ii) and (iii) (this expression vector is also referred to as a secretory expression vector): (I) promoter DNA, (Ii) The following: (A) a peptide comprising the amino acid sequence represented by SEQ ID NO: 2, (B) a peptide having an amino acid sequence having at least 70% sequence identity with the amino acid sequence represented by SEQ ID NO: 2 and having a secretion signal activity; (C) a peptide having an amino acid sequence obtained by substituting, deleting or adding one or several amino acid residues to the amino acid sequence represented by SEQ ID NO: 2, and having a secretion signal activity; (D) a peptide having a secretory signal activity encoded by a base sequence having at least 70% sequence identity with the base sequence represented by SEQ ID NO: 1, and (e) a base sequence represented by SEQ ID NO: 1.
- a peptide encoded by a base sequence that hybridizes with a complementary strand of DNA and having a secretion signal activity A DNA encoding any peptide selected from the group consisting of: and (iii) a DNA encoding the target protein, or a cloning site for inserting a DNA encoding the target protein.
- the promoter is a SED1 promoter.
- (iii) is DNA encoding a target protein.
- the present invention provides a transformed yeast into which the secretory expression vector is introduced.
- the present invention provides a method for producing a yeast that secrete-produces a protein, the method comprising the following: A promoter DNA; (a) a peptide comprising the amino acid sequence represented by SEQ ID NO: 2, (b) an amino acid sequence having at least 70% sequence identity with the amino acid sequence represented by SEQ ID NO: 2, and secretory signal activity (C) a peptide having an amino acid sequence obtained by substituting, deleting or adding one or several amino acid residues to the amino acid sequence represented by SEQ ID NO: 2, and having a secretion signal activity (D) a peptide encoded by a base sequence having at least 70% sequence identity with the base sequence represented by SEQ ID NO: 1 and having a secretory signal activity, and (e) a base sequence represented by SEQ ID NO: 1.
- the present invention provides a method for secreting and producing a protein in yeast, and this method includes a step of culturing the transformed yeast or the yeast produced by the method.
- the present invention provides an expression vector comprising the following (i), (ii), (iii) and (iv) (this expression vector is also referred to as a surface display type expression vector): (I) promoter DNA, (Ii) The following: (A) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2; (B) a peptide having an amino acid sequence having at least 70% sequence identity with the amino acid sequence represented by SEQ ID NO: 2 and having a secretion signal activity; (C) a peptide having an amino acid sequence obtained by substituting, deleting or adding one or several amino acid residues to the amino acid sequence represented by SEQ ID NO: 2, and having a secretion signal activity; (D) a peptide having a secretory signal activity encoded by a base sequence having at least 70% sequence identity with the base sequence represented by SEQ ID NO: 1, and (e) a base sequence represented by SEQ ID NO: 1.
- a peptide encoded by a base sequence that hybridizes with a complementary strand of DNA and having a secretion signal activity DNA encoding any peptide selected from the group consisting of: (Iii) DNA encoding the target protein, or a cloning site for inserting DNA encoding the target protein; and (Iv) DNA encoding an anchor domain.
- the promoter is a SED1 promoter.
- the anchor domain is a SED1 anchor domain.
- the above (iii) is DNA encoding a target protein.
- the present invention provides a transformed yeast into which the expression vector of the surface display type is introduced.
- the present invention provides a method for producing a yeast that displays proteins on the surface, which method comprises the following: A promoter DNA; (a) a peptide comprising the amino acid sequence represented by SEQ ID NO: 2, (b) an amino acid sequence having at least 70% sequence identity with the amino acid sequence represented by SEQ ID NO: 2, and secretory signal activity (C) a peptide having an amino acid sequence obtained by substituting, deleting or adding one or several amino acid residues to the amino acid sequence represented by SEQ ID NO: 2, and having a secretion signal activity (D) a peptide encoded by a base sequence having at least 70% sequence identity with the base sequence represented by SEQ ID NO: 1 and having a secretory signal activity, and (e) a base sequence represented by SEQ ID NO: 1.
- proteins can be secreted or displayed on the cell surface with high activity.
- the secretory signal peptide of the present invention can be suitably used for both protein secretion and cell surface display. Furthermore, the secretion signal peptide of the present invention can stably impart high secretion ability in combination with various promoters and protein genes.
- 6 is a graph showing changes over time in ⁇ -glucosidase activity of bacterial cells during culture for various surface-displayed transformed yeasts obtained using expression cassettes X1 to X4.
- 6 is a graph showing the time course of ⁇ -glucosidase activity in the medium during culture for various secretory transformed yeasts obtained using expression cassettes X5 to X8.
- 6 is a graph showing endoglucanase activity of cells after 48 hours of culturing for various surface-displayed transformed yeasts obtained using expression cassettes X9 to X11.
- 6 is a graph showing changes over time in ⁇ -glucosidase activity of bacterial cells during culture for various surface-displayed transformed yeasts obtained using expression cassettes X12 to X17.
- 6 is a graph showing changes over time in ⁇ -glucosidase activity in a medium during culture for various secretory transformed yeasts obtained using expression cassettes X18 and X19. 6 is a graph showing the relative value of GFP fluorescence intensity in the medium after 24 hours of culturing for various secretory transformed yeasts obtained using expression cassettes X20 to X22.
- a secretory signal peptide is a peptide that is usually bound to the N-terminus of a protein that is localized in the cell membrane, cell wall, or secreted outside the cell membrane.
- the secretory signal peptide is usually removed by being degraded by a signal peptidase in the process of secreting a secreted protein from the cell through the cell membrane and secreted outside the cell.
- a secretory signal peptide is linked to the N-terminus of a protein (secretory protein) or a heterologous protein that originally has the secretory signal peptide by linking these proteins in the cell in which the protein is to be expressed, Functions to secrete heterologous proteins out of the cell.
- secretory signal activity functioning as a secretory signal peptide in expression of a target protein in yeast is also referred to as “secretory signal activity”.
- any peptide selected from the group consisting of the following (a) to (e) is provided as a secretory signal peptide: (A) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2; (B) a peptide having an amino acid sequence having at least 70% sequence identity with the amino acid sequence represented by SEQ ID NO: 2 and having a secretory signal activity; (C) a peptide having an amino acid sequence obtained by substituting, deleting or adding one or several amino acid residues to the amino acid sequence represented by SEQ ID NO: 2 and having a secretion signal activity; (D) a peptide having a secretory signal activity encoded by a base sequence having at least 70% sequence identity with the base sequence represented by SEQ ID NO: 1; and (e) a base sequence represented by SEQ ID NO: 1.
- a DNA encoding any peptide selected from the group consisting of the above (a) to (e) (herein, also simply referred to as “secretory signal peptide of the present invention”)
- a polynucleotide comprising is also provided.
- An example of DNA encoding the secretory signal peptide of the present invention is the base sequence represented by SEQ ID NO: 1, which codes for the amino acid sequence represented by SEQ ID NO: 2.
- the amino acid sequence represented by SEQ ID NO: 2 and the base sequence encoding the amino acid sequence (SEQ ID NO: 1) are the secretory signal peptides of SED1, which is a major cell surface localized protein in the stationary phase of the yeast Saccharomyces cerevisiae. Although derived, the origin is not limited to this. SED1 is induced by stress and is thought to contribute to the maintenance of cell wall integrity.
- the gene of SED1 (Sed1) can be obtained, for example, using a method commonly used by those skilled in the art based on sequence information registered in GenBank (GenBank accession number NM_001180385; NCBI Gene ID: 851649).
- the protein or peptide described in the present specification consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the disclosed amino acid sequence, and has a desired function or effect in the present invention.
- the protein or peptide may be substantially present, and the polynucleotide may include one that encodes such a protein or peptide. Any one type of amino acid mutation (for example, deletion, substitution or addition) relative to the disclosed amino acid sequence may be used, or two or more types may be combined. The total number of these mutations is 1 or several, but is not particularly limited as long as it substantially has the desired function or effect, and may depend on the size of the protein or peptide.
- the total number of mutations is, for example, 1 or more and 10 or less, 1 or more and 5 or less, 1 or more and 4 or less, 1 or more and 3 or less, or 1 or more and 2 or less, but is not limited thereto.
- the total number of mutations can be within a range that satisfies the sequence identity described below, for example.
- Examples of amino acid substitution may be any substitution as long as each function or effect is substantially retained. For example, conservative substitution can be mentioned.
- conservative substitution include substitution within the following groups (ie, between amino acids shown in parentheses): (glycine, alanine), (valine, isoleucine, leucine), (aspartic acid, glutamic acid) ), (Asparagine, glutamine), (serine, threonine), (lysine, arginine), (phenylalanine, tyrosine).
- the protein or peptide described herein has an amino acid sequence that has, for example, 70% or more sequence identity to the disclosed amino acid sequence and is desired in the present invention. Or a polynucleotide that encodes such a protein or peptide. Sequence identity in the amino acid sequence can also be 74% or more, 78% or more, 80% or more, 85% or more, 90% or more, 92% or more, 95% or more, 98% or more, or 99% or more.
- sequence identity or similarity is a relationship between two or more proteins or two or more polynucleotides determined by comparing sequences, as is known in the art. .
- Sequence “identity” means between protein or polynucleotide sequences, as determined by alignment between protein or polynucleotide sequences, or in some cases by alignment between a series of partial sequences. Means the degree of sequence invariance.
- similarity refers to a protein or polynucleotide sequence as determined by alignment between protein or polynucleotide sequences, or in some cases by alignment between a series of partial sequences. It means the degree of correlation.
- the method for determining identity and similarity is preferably a method designed to align the longest between the sequences to be compared. Methods for determining identity and similarity are provided as programs available to the public. For example, the BLAST (Basic Local Alignment Search Tool) program by Altschul et al. (Eg Altschul et al., J. Mol. Biol., 1990, 215: 403-410; Altschyl et al, Nucleic Acids Res., 1997, 25: 3389-3402 ) Can be determined.
- the conditions for using software such as BLAST are not particularly limited, but it is preferable to use default values.
- the protein or peptide described herein may be encoded by DNA hybridized with DNA comprising a base sequence complementary to the DNA comprising the disclosed base sequence.
- the polynucleotide may comprise such hybridized DNA.
- the hybridization between the DNA comprising the disclosed base sequence and the DNA comprising a complementary base sequence is preferably carried out under stringent conditions.
- the stringent condition refers to, for example, a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed.
- a nucleic acid having high nucleotide sequence identity that is, a disclosed nucleotide sequence, for example, 70% or more, 75% or more, 78% or more, 80% or more, 85% or more, 90% or more, 92% or more, 95%
- a condition in which a complementary strand of a DNA comprising a base sequence having 98% or more identity or 99% or more identity is hybridized and a complementary strand of a nucleic acid having a lower homology is not hybridized.
- the sodium salt concentration is, for example, 15 mM to 750 mM, 50 mM to 750 mM, or 300 mM to 750 mM
- the temperature is, for example, 25 ° C. to 70 ° C., 50 ° C. to 70 ° C., or 55 ° C. to 65 ° C.
- the concentration is 0% to 50%, 20% to 50%, or 35% to 45%.
- the filter washing conditions after hybridization are such that the sodium salt concentration is, for example, 15 mM to 600 mM, 50 mM to 600 mM, or 300 mM to 600 mM, and the temperature is, for example, 50 ° C.
- the DNA that hybridizes under stringent conditions is, for example, after hybridization at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which the DNA is immobilized.
- Examples include DNA that can be obtained by washing the filter at 65 ° C. in an SSC solution having a concentration of 0.1 to 2 times (the composition of the SSC solution having a concentration of 1 is 150 mM NaCl and 15 mM sodium citrate).
- Hybridization can be performed by a known method such as the method described in Sambrook et al., Molecular® Cloning, A • Laboratory • Manual, 3rd • Ed ,, • Cold • Spring • Harbor • Laboratory (2001). The higher the temperature or the lower the salt concentration, the higher the stringency, and a polynucleotide with higher homology (sequence identity) can be isolated.
- the polynucleotides described herein may have the disclosed base sequences and, for example, 70% or more, 75% or more, 78% or more, 80% or more, 85% or more, 90% or more, Examples thereof include polynucleotides having a base sequence having an identity of 92% or more, 95% or more, 98% or more, or 99% or more and substantially having the desired function or effect.
- a base sequence encoding a predetermined amino acid sequence (for example, the amino acid sequence represented by SEQ ID NO: 2) encodes the predetermined amino acid sequence without changing the amino acid sequence of the protein by substitution based on the degeneracy of the genetic code. At least one base in the base sequence can be replaced with another base.
- the DNA encoding the secretory signal peptide of the present invention also includes DNA having a base sequence altered by substitution based on the degeneracy of the genetic code.
- the DNA encoding the secretory signal peptide of the present invention or the polynucleotide containing the DNA is, for example, a predetermined yeast (for example, Saccharomyces cerevisiae) using a primer designed based on the base sequence represented by SEQ ID NO: 1. It can be obtained as a nucleic acid fragment by performing PCR using DNA extracted from the above, nucleic acids derived from various cDNA libraries or genomic DNA libraries as a template.
- the DNA encoding the secretory signal peptide of the present invention or the polynucleotide containing the DNA is hybridized to the nucleic acid derived from the library using a probe designed based on the nucleotide sequence represented by SEQ ID NO: 1.
- the DNA encoding the secretory signal peptide of the present invention or the polynucleotide containing the DNA may be synthesized as a nucleic acid fragment by various nucleic acid sequence synthesis methods known in the art such as chemical synthesis methods.
- the DNA encoding the secretory signal peptide of the present invention is, for example, a DNA comprising the base sequence represented by SEQ ID NO: 1, a conventional mutagenesis method, site It can be obtained by modification by a specific mutation method, a molecular evolution method using error-prone PCR, or the like.
- a known method such as the Kunkel method or the Gapped-duplex method or a method equivalent thereto can be cited.
- a mutagenesis kit for example, Mutant-K (TAKARA), Mutant-G (TAKARA), etc.
- TAKARA LA PCR in vitro Mutations are introduced using the Mutagenesis series kit.
- a secretion cassette for secreting and producing the target protein and a surface layer display for displaying the target protein on the cell surface Expression cassettes such as cassettes can be made.
- the surface display cassette contains DNA encoding the anchor domain.
- Cell surface localized protein refers to a protein that is immobilized on, attached to, or adhered to the cell surface and is localized there. Proteins modified with lipids are known as cell surface localized proteins, and these lipids are immobilized on cell membranes by covalent bonding with membrane components.
- GPI glycosyl phosphatidyl inositol: ethanolamine phosphate-6 mannose ⁇ -1,2 mannose ⁇ -1,6 mannose ⁇ -1,4 glucosamine ⁇ -1,6 inositol phospholipid A glycolipid having a basic structure
- the GPI anchor protein has GPI which is a glycolipid at its C-terminal, and this GPI is bound to the cell membrane surface by covalently binding to PI (phosphatidyl tinositol) in the cell membrane.
- GPI binding to the C-terminus of the GPI anchor protein is performed as follows.
- the GPI anchor protein is secreted into the endoplasmic reticulum lumen by the action of a secretory signal present on the N-terminal side after transcription and translation.
- a region called a GPI anchor attachment signal that is recognized when the GPI anchor binds to the GPI anchor protein is present at the C-terminal of the GPI anchor protein or in the vicinity thereof.
- this GPI anchor attachment signal region is cleaved, and GPI binds to the newly generated C-terminus.
- the protein to which GPI is bound is transported to the cell membrane by secretory vesicles, and is fixed to the cell membrane by GPI covalently binding to PI of the cell membrane. Further, the GPI anchor is cleaved by phosphatidylinositol-dependent phospholipase C (PI-PLC), and is incorporated into the cell wall so that it is fixed to the cell wall and presented on the cell surface.
- PI-PLC phosphatidylinositol-dependent phospholipase C
- a polynucleotide encoding the entire GPI anchor protein, which is a cell surface localized protein, or a region containing a GPI anchor adhesion signal region, which is a cell membrane binding region can be used.
- the cell membrane binding region (GPI anchor attachment signal region) is usually a region on the C-terminal side of the cell surface localized protein.
- the cell membrane binding region only needs to contain a GPI anchor attachment signal region, and may contain any other part of the GPI anchor protein as long as the enzyme activity of the fusion protein is not inhibited.
- the GPI anchor protein may be any protein that functions in yeast cells.
- GPI anchor proteins include ⁇ - or a-agglutinin (AG ⁇ 1, AGA1), TIP1, FLO1, SED1, CWP1, and CWP2.
- SED1 or a cell membrane binding region thereof is used.
- the base sequence of the anchor protein coding region of Sed1 is shown in SEQ ID NO: 4, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 5 (provided that SEQ ID NOs: 4 and 5 respectively represent the start codon and the methionine encoded thereby. Not included).
- the cell membrane binding region (GPI anchor attachment signal region) of SED1 is a region including, for example, positions 109 to 337 of SEQ ID NO: 5.
- the SED1 anchor domain even if it is the full length of the amino acid sequence of SEQ ID NO: 5 or unless the anchor function is impaired, a partial sequence thereof (for example, a sequence comprising the amino acid sequence of positions 109 to 337 of SEQ ID NO: 5) ).
- sequence identity and hybridization conditions described above apply to the anchor domain and its coding DNA.
- DNA encoding an anchor domain is known using DNA extracted from microorganisms having these, nucleic acids derived from various cDNA libraries or genomic DNA libraries as templates. It can be obtained as a nucleic acid fragment by PCR using primers designed based on the sequence information. Such a polynucleotide can be obtained as a nucleic acid fragment by performing hybridization on a nucleic acid derived from the library using a probe designed based on known sequence information. It can also be cut out as a nucleic acid fragment from an existing vector containing the polynucleotide. Polynucleotides may be synthesized as nucleic acid fragments by various nucleic acid sequence synthesis methods known in the art, such as chemical synthesis methods.
- Promoter DNA The promoter DNA only needs to have promoter activity, and “promoter activity” refers to an activity that causes transcription factors to bind to the promoter region and cause transcription.
- Promoter DNA can be excised from bacterial cells, phages, and the like that possess the desired promoter region using restriction enzymes. If necessary, a DNA fragment of the promoter region can be obtained by amplifying a desired promoter region by PCR using a primer provided with a restriction enzyme recognition site or an overlapping site with a cloning vector. Further, a desired promoter DNA may be chemically synthesized based on the already known base sequence information of the promoter region.
- the promoter DNA contained in the polynucleotide of the present invention can be any promoter as long as it has promoter activity in yeast.
- the promoter DNA may be that of the gene itself intended for expression or may be derived from another gene. Further, it may be a promoter of a gene encoding a cell surface localized protein used as an anchor domain. Examples include SED1 promoter, TDH3 promoter, PGK1 promoter, CWP2 promoter, and TDH1 promoter.
- the base sequences of these promoters are the base sequences represented by SEQ ID NOs: 3, 12, 13, 18, and 21, respectively.
- nucleotide sequence is a nucleotide sequence in which one or more (for example, several) nucleotides are mutated such as deleted, added, or substituted. Good.
- target protein The type of the target protein or its origin is not particularly limited.
- the target protein include enzymes, antibodies, ligands, and fluorescent proteins.
- the enzyme include cellulose degrading enzyme, starch degrading enzyme, glycogen degrading enzyme, xylan degrading enzyme, chitin degrading enzyme, lipid degrading enzyme and the like. More specifically, for example, endoglucanase, cellobiohydrolase, and Examples include ⁇ -glucosidase, amylase (for example, glucoamylase and ⁇ -amylase), lipase and the like.
- the DNA encoding the target protein is preferably a cDNA sequence excluding introns.
- the DNA encoding the target protein may be a sequence encoding the entire length thereof, or may be a sequence encoding a partial region of the target protein as long as it exhibits the activity of the target protein.
- the base sequence encoding the natural protein contains one or more (for example, several) nucleotides including mutations such as deletion, addition or substitution.
- it may be a base sequence encoding a protein consisting of an amino acid sequence containing a mutation such as deletion, addition or substitution of one or more (for example, several) amino acids.
- the DNA (gene) encoding the target protein was designed based on known sequence information using DNA extracted from microorganisms having or producing the protein, nucleic acids derived from various cDNA libraries or genomic DNA libraries as templates. It can be obtained as a nucleic acid fragment by PCR using primers. Such a polynucleotide can be obtained as a nucleic acid fragment by performing hybridization on a nucleic acid derived from the library using a probe designed based on known sequence information. Moreover, it can also be cut out from an existing vector containing DNA encoding the target protein and used as a nucleic acid fragment (may be in the form of an expression cassette). Such a gene can also be obtained by artificial synthesis based on a sequence optimized in consideration of the codon frequency of the host as necessary.
- cellulolytic enzyme As an example of the target protein, cellulolytic enzyme will be described below as an example.
- Cellulolytic enzyme refers to any enzyme capable of cleaving ⁇ 1,4-glycoside bonds.
- the cellulolytic enzyme can be derived from any cellulose hydrolase producing bacterium.
- Cellulose hydrolase producing bacteria typically include the genus Aspergillus (for example, Aspergillus aculeatus, Aspergillus niger, Aspergillus oryzae), Trichoderma genus (for example, Trichoderma reesei), Clostridium genus (eg Clostridium thermocellum), Cellulomonas genus (eg Cellulomonas fimi and Cellulomonas uda), Examples include microorganisms belonging to the genus Pseudomonas (for example, Pseudomonas fluorescence).
- endoglucanase cellobiohydrolase
- ⁇ -glucosidase will be described as typical cellulolytic enzymes, but the cellulolytic enzymes are not limited to these.
- Endoglucanase is an enzyme usually called cellulase, which cleaves cellulose from the inside of the molecule to produce glucose, cellobiose, and cellooligosaccharide (“cellulose intramolecular cleavage”).
- cellulase There are five types of endoglucanases, referred to as endoglucanase I, endoglucanase II, endoglucanase III, endoglucanase IV, and endoglucanase V, respectively. These distinctions are differences in amino acid sequences, but are common in that they have a cellulose intramolecular cleavage action.
- endoglucanase derived from Trichoderma reesei particularly, endoglucanase II: EGII (for example, Patent Document 6) can be used, but is not limited thereto.
- Cellobiohydrolase decomposes from either the reducing end or the non-reducing end of cellulose to release cellobiose (“terminal cleavage of cellulose molecule”).
- cellobiohydrolase derived from Trichoderma reesei particularly, cellobiohydrolase II: CBHII (for example, Patent Document 6)
- CBHII for example, Patent Document 6
- ⁇ -Glucosidase is an exo-type hydrolase that cleaves glucose units from the non-reducing end of cellulose (“glucose unit cleavage”).
- ⁇ -glucosidase can cleave ⁇ 1,4-glycosidic bond between aglycone or sugar chain and ⁇ -D-glucose, and can hydrolyze cellobiose or cellooligosaccharide to produce glucose.
- ⁇ -glucosidase is a representative example of an enzyme capable of hydrolyzing cellobiose or cellooligosaccharide.
- One type of ⁇ -glucosidase is currently known and is referred to as ⁇ -glucosidase 1.
- ⁇ -glucosidase (particularly ⁇ -glucosidase 1: BGL1 (for example, Non-Patent Document 7)) derived from Aspergillus acreatas can be used, but is not limited thereto.
- enzymes having different cellulose hydrolysis modes may be combined. Enzymes that act in a variety of different cellulose hydrolysis modes such as cellulose intramolecular cleavage, cellulose molecular end cleavage, and glucose unit cleavage can be combined as appropriate. Examples of enzymes having each hydrolysis mode include, but are not limited to, endoglucanase, cellobiohydrolase, and ⁇ -glucosidase. The combination of enzymes with different modes of cellulose hydrolysis can be selected, for example, from the group consisting of endoglucanase, cellobiohydrolase, and ⁇ -glucosidase.
- glucose which is a constituent sugar of cellulose
- an enzyme capable of producing glucose in addition to a glucose unit cleaving enzyme (for example, ⁇ -glucosidase), endoglucanase can also produce glucose.
- ⁇ -glucosidase in yeast, ⁇ -glucosidase, endoglucanase, and cellobiohydrolase can be secreted or displayed on the surface.
- the polynucleotide containing the secretion cassette or the surface display cassette can further contain a terminator DNA.
- the terminator DNA only needs to have terminator activity, and “terminator activity” refers to the activity of terminating transcription in the terminator region.
- the terminator only needs to have terminator activity, and can be excised from bacterial cells, phages, and the like possessing a desired terminator region using a restriction enzyme.
- a DNA fragment of the terminator region can be obtained by amplifying a desired terminator region by PCR using a primer provided with a restriction enzyme recognition site or a cloning vector insertion site as necessary.
- a desired terminator DNA may be chemically synthesized based on the already known base sequence information of the terminator region.
- Examples of the terminator DNA include ⁇ -agglutinin terminator, ADH1 (aldehyde dehydrogenase) terminator, TDH3 (glyceraldehyde-3'-phosphate dehydrogenase) terminator, DIT1 terminator and the like.
- an “expression cassette” means that a DNA encoding a target protein and various regulatory elements for controlling the expression thereof are contained in a host microorganism or cell so that the target protein can be expressed.
- a DNA sequence or polynucleotide that is operably linked refers to a DNA sequence or polynucleotide that is operably linked.
- operably linked means an expression cassette so that the DNA encoding the protein of interest is expressed under the control of a promoter and optionally under the control of other regulatory elements.
- each component contained in the expression vector is linked.
- Each component may be further linked with a sequence such as a linker between the components as long as the target protein can be expressed.
- expression cassettes include secretory expression cassettes and surface layer presentation expression cassettes as described below.
- the secretory expression cassette includes promoter DNA, DNA encoding the secretory signal peptide of the present invention, and DNA encoding the target protein (this expression cassette is also referred to as a secretion cassette).
- the surface display type expression cassette includes promoter DNA, DNA encoding the secretory signal peptide of the present invention, DNA encoding the target protein, and DNA encoding the anchor domain (this expression cassette is also referred to as surface display cassette). .
- the promoter DNA, the DNA encoding the secretion signal peptide, and the DNA encoding the target protein can be ligated in a state capable of functioning in the host microorganism or host cell.
- the secretion cassette is constructed, for example, from 5 ′ to 3 ′ so as to contain a promoter DNA, a DNA encoding the secretion signal peptide of the present invention, and a DNA encoding the target protein in the order described.
- the secretion cassette may further contain a terminator DNA downstream of the DNA encoding the protein of interest.
- the promoter DNA, the DNA encoding the secretory signal peptide, the DNA encoding the target protein, and the DNA encoding the anchor domain can be linked in a state capable of functioning in the host microorganism or host cell.
- the surface layer display cassette includes, for example, from 5 ′ to 3 ′, promoter DNA, DNA encoding the secretory signal peptide of the present invention, DNA encoding the target protein, and DNA encoding the anchor domain in the order described. Built in.
- the surface display cassette may further contain a terminator DNA downstream of the DNA encoding the anchor domain.
- Synthesis and binding of DNA containing various sequences can be performed by techniques that can be commonly used by those skilled in the art.
- the DNA of each constituent element provided with an appropriate restriction enzyme recognition sequence by PCR is cleaved with the restriction enzyme and ligated using ligase or the like, or One-step It can be performed using isothermal assembly (Non-patent Document 8).
- Non-patent Document 8 it is possible to cleave the secretory signal peptide accurately and to express the active enzyme.
- a plasmid containing a region (structural gene) encoding a target protein for example, an enzyme such as endoglucanase, cellobiohydrolase, ⁇ -glucosidase, fluorescent protein, or various antibodies
- a target protein for example, an enzyme such as endoglucanase, cellobiohydrolase, ⁇ -glucosidase, fluorescent protein, or various antibodies
- an expression regulatory sequence such as a promoter and terminator
- the expression cassette (secretory cassette and surface display cassette) of the present invention may contain a cloning site for inserting DNA encoding the target protein instead of including DNA encoding the target protein.
- cloning sites are known in the art, and for example, multiple cloning sites containing sites recognized by various restriction enzymes can be utilized. In the case of an expression cassette containing such a cloning site, it becomes easy to insert DNA encoding the target protein into the expression cassette via the cloning site.
- the present invention provides an expression vector comprising the above expression cassette.
- the secretory expression vector of the present invention includes the secretory expression cassette, and the surface display type expression vector of the present invention includes the surface display type expression cassette.
- the “expression vector” refers to a vector into which a unit (expression cassette) for expressing a DNA encoding a target protein is inserted, and includes a vector into which a DNA encoding the target protein is inserted.
- the expression vector may be a plasmid vector or an artificial chromosome. When yeast is used as a host, the form of a plasmid is preferable because the preparation of a vector is easy and the transformation of yeast cells is easy.
- the vector can include regulatory sequences (operators, enhancers, etc.).
- Such vectors have, for example, a yeast 2 ⁇ m plasmid replication origin (Ori) and a ColE1 replication origin, and yeast selection markers (described below) and E. coli selection markers (such as drug resistance genes). ).
- auxotrophic marker gene eg, gene encoding imidazoleglycerol phosphate dehydrogenase (HIS3), gene encoding malate beta-isopropyl dehydrogenase (LEU2), O-acetylhomoserine O-acetylserine sulfide
- MET15 gene encoding tryptophan synthase
- ARG4 gene encoding argininosuccinate lyase
- TRP1 Gene encoding histidinol dehydrogenase (HIS4)
- gene encoding orotidine-5-phosphate decarboxylase (URA3) dihydroorotic acid dehydrogenase
- UAA3 dihydroorotic acid dehydrogenase
- ZE dihydroorotic acid dehydrogenase
- the yeast used as the host is not particularly limited as long as it belongs to Ascomycetous yeast.
- Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Kloeckera Examples include yeasts such as the genus Schwanniomyces, the genus Komagataella, and the genus Yarrowia.
- the yeast of the present invention can be obtained by introducing the above expression cassette or expression vector into a yeast serving as a host.
- “Introduction” includes not only introduction of a gene (DNA encoding a target protein) for expression in an expression cassette or expression vector into the host cell, but also expression in the host cell.
- the introduction method is not particularly limited, and a known method can be adopted.
- a typical example is a method of transforming yeast using the expression vector of the present invention described above. Transformation methods are not particularly limited, and known methods such as transfection methods such as calcium phosphate method, electroporation method, lipofection method, DEAE dextran method, lithium acetate method, protoplast method, and microinjection method are used without limitation. it can.
- the introduced gene may exist in the form of a plasmid, or may exist in a form inserted into a yeast chromosome or in a form integrated into a yeast chromosome by homologous recombination.
- a yeast that secretes proteins can be produced by introducing an expression vector containing a secretion cassette into yeast and obtaining transformed yeast.
- yeast that presents proteins on the cell surface layer can be prepared.
- the yeast into which the above expression cassette or expression vector has been introduced is characterized by the characteristics of the yeast selection marker, the target protein activity of the bacterial cell (surface display type), or the activity of the target protein outside the bacterial cell (secretory type) according to conventional methods. It can be selected as an indicator.
- the target protein is immobilized (cell surface display) on the cell surface of the transformed yeast obtained by introducing an expression vector containing the surface layer display cassette into the yeast.
- a fluorescent labeled streptavidin is allowed to act after the reaction.
- each expression vector including a gene expression cassette having a sequence encoding each of a plurality of types of proteins may be constructed, or a plurality of gene expression cassettes may be put into one expression vector.
- a vector for simultaneous expression of three genes pATP403 can be used (Non-patent Document 9).
- the transformed yeast of the present invention can be cultured under culture conditions generally applicable to yeast.
- Culture of the transformed yeast can be appropriately performed by methods well known to those skilled in the art.
- the medium composition, culture pH and culture temperature can be appropriately set according to the properties of the yeast and the target protein.
- the cell density and the culture time during the culture can also be appropriately set according to the properties of the yeast and the target protein.
- a method for secreting and producing a protein is also provided.
- This method can be carried out using the above-described culture step for secretory transformed yeast.
- proteins such as antibodies and enzymes can be secreted and produced by this method.
- a step of recovering the production-containing fraction from the culture solution, and a step of purifying or concentrating it can also be performed.
- culture conditions generally applied to yeast can be appropriately selected and used.
- stationary culture, shaking culture, aeration-agitation culture, or the like can be used for culture for fermentation.
- the aeration conditions can be appropriately selected from anaerobic conditions, microaerobic conditions, aerobic conditions, and the like.
- the medium composition, medium pH, culture temperature, cell density and culture time during culture, and subsequent collection, purification, and concentration can be appropriately set.
- amino acid sequences of the SED1 secretion signal (SEQ ID NO: 2) and the CWP2 secretion signal (SEQ ID NO: 20) are obtained from the signal peptide prediction program PSORT (http: // psort. nibb.ac.jp/) (available as WoLF PSORT (http://www.genscript.com/psort/wolf_psort.html)).
- Non-Patent Document 8 of the yeast Saccharomyces cerevisiae used in this example was obtained from Invitrogen.
- the gene transfer to all yeasts shown in this example was performed by the lithium acetate method.
- Plasmids containing the following expression cassettes X1 to X19 were prepared: X1: SED1 promoter + glucoamylase secretion signal + BGL1 + SED1 anchor domain X2: SED1 promoter + SED1 secretion signal + BGL1 + SED1 anchor domain X3: SED1 promoter + MF ⁇ prepro + BGL1 + SED1 anchor domain X4: SED1 promoter + HKR1 secretion signal + BGL1 + SED1 anchor domain + GL1 + GL GL X6: SED1 promoter + SED1 secretion signal + BGL1 X7: SED1 promoter + MF ⁇ prepro + BGL1 X8: SED1 promoter + HKR1 secretion signal + BGL1 X9: SED1 promoter + glucoamylase secretion signal + EGII + SED1 anchor domain X
- SEQ ID NO: 1 nucleotide sequence of DNA encoding SED1 secretion signal peptide derived from Saccharomyces cerevisiae
- SEQ ID NO: 2 amino acid sequence of SED1 secretion signal peptide derived from Saccharomyces cerevisiae
- SEQ ID NO: 3 SED1 promoter derived from Saccharomyces cerevisiae
- SEQ ID NO: 4 DNA sequence of the region of the SED1 anchor protein derived from Saccharomyces cerevisiae used as the SED1 anchor domain in the examples of the present invention except the start codon
- SEQ ID NO: 5 Amino acid sequence of SED1 anchor protein derived from Saccharomyces cerevisiae (but not including initiating methionine) used as SED1 anchor domain in the examples
- SEQ ID NO: 6 derived from Rhizopus oryzae
- SEQ ID NO: 7 of the DNA encoding the leco
- the Saccharomyces cerevisiae-derived cell surface localized protein gene Sed1 is obtained by PCR using the Saccharomyces cerevisiae BY4741 strain genome as a template and the primer pair Sed1p-F (SEQ ID NO: 24) and Sed1ss-R (SEQ ID NO: 25). Amplified to prepare a DNA fragment containing a promoter region and a secretory signal sequence.
- This fragment was transformed into vector plasmid pIBG-SGS (auxotrophic marker gene HIS3, and BGL1 expression cassette (ie, SED1 promoter, secretory signal peptide sequence of Rhizopus oryzae-derived glucoamylase, ⁇ -glucosidase 1 derived from Aspergillus acreatas (BGL1)).
- a fragment containing the MF ⁇ prepro leader sequence derived from Saccharomyces cerevisiae was subjected to pIUPGSBAAG (MF ⁇ prepro sequence and ⁇ -amylase having the 3 ′ half region of the ⁇ -agglutinin gene by PCR): Non-patent literature 11) was used as a template, and it was prepared by amplification using primer pair MFa-F (SEQ ID NO: 28) and MFA-R (SEQ ID NO: 29). This fragment was ligated to the fragment amplified using the vector plasmid pIBG-SGS using the primer pair BGL1-F2 (SEQ ID NO: 30) and Sed1p-R (SEQ ID NO: 31) by One-step isothermal assembly.
- the obtained plasmid containing the expression cassette X3 was named pIBG-SMS.
- the secreted protein gene Hkr1 derived from Saccharomyces cerevisiae was amplified by PCR using the Saccharomyces cerevisiae BY4741 genome as a template and using a primer pair Hkr1ss-F (SEQ ID NO: 32) and Hkr1ss-R (SEQ ID NO: 33).
- a DNA fragment containing a secretory signal sequence was prepared. This fragment was ligated to the fragment amplified using the vector plasmid pIBG-SGS using the primer pair BGL1-F3 (SEQ ID NO: 34) and Sed1p-R2 (SEQ ID NO: 35) by One-step isothermal assembly.
- the resulting plasmid containing the expression cassette X4 was named pIBG-SHS.
- a sequence excluding the coding region of the SED1 gene of pIBG-SGS, pIBG-SSS, pIBG-SMS, and pIBG-SHS was used as a template for each of the primer pairs PRS-BsrGI-F (SEQ ID NO: 36) and BGL1- It was prepared by amplification using BsrGI-R (SEQ ID NO: 37). Each of these fragments was treated with BsrGI and circularized by the self-ligation method.
- the obtained plasmids containing the expression cassette X5, X6, X7, or X8 were named pIBG-SGsec, pIBG-SSsec, pIBG-SMsec, and pIBG-SHsec, respectively.
- Trichoderma reesei-derived endoglucanase II (EGII) gene was subjected to pIEG-SGS (auxotrophic marker gene HIS3 and EGII expression cassette (ie, SED1 promoter, Rhizopus oryzae-derived glucoamylase secretion signal) by PCR.
- pIEG-SGS auxotrophic marker gene HIS3 and EGII expression cassette (ie, SED1 promoter, Rhizopus oryzae-derived glucoamylase secretion signal) by PCR.
- Non-patent It was prepared by amplifying using primer pair EGII-F (SEQ ID NO: 38) and EGII-R (SEQ ID NO: 39) using the template (equivalent to pIEG-SS in Reference 10) as a template.
- This fragment was ligated to the fragment amplified using the vector plasmid pIBG-SSS using the primer pair Sed1a-F (SEQ ID NO: 40) and Sed1ss-R2 (SEQ ID NO: 41) by One-step isothermal assembly.
- the obtained plasmid containing the expression cassette X10 was named pIEG-SSS.
- the coding region of the EGII gene was prepared by amplifying by PCR using pIEG-SGS as a template and using a primer pair EGII-F2 (SEQ ID NO: 42) and EGII-R (SEQ ID NO: 39). This fragment was ligated to the fragment amplified using the vector plasmid pIBG-SMS as a template and using the primer pair Sed1a-F (SEQ ID NO: 40) and MFa-R2 (SEQ ID NO: 43) by One-step isothermal assembly.
- the obtained plasmid containing the expression cassette X11 was named pIEG-SMS.
- the promoter region of the gene TDH3 derived from Saccharomyces cerevisiae was subjected to pIBG-TGS (auxotrophic marker gene HIS3 and BGL1 expression cassette (ie, TDH3 promoter, secretory signal peptide sequence of Rhizopus oryzae-derived glucoamylase, BGL1) by PCR.
- This fragment was ligated to the fragment amplified with the primer pair Sed1ss-F (SEQ ID NO: 46) and PRS-R2 (SEQ ID NO: 47) using the vector plasmid pIBG-SSS as a template by One-step isothermal assembly.
- the obtained plasmid containing the expression cassette X13 was named pIBG-TSS.
- the promoter region of the gene PGK1 derived from Saccharomyces cerevisiae was subjected to PCR using pGK403 (protein expression vector having a promoter region and a terminator region of PGK1: non-patent document 12) as a template and a primer pair PGK1p-F (SEQ ID NO: 50). ) And PGK1p-R (SEQ ID NO: 51).
- This fragment was ligated to the fragment amplified by using the vector plasmid pIBG-SGS as a template and the primer pair GAss-F (SEQ ID NO: 52) and PRS-R3 (SEQ ID NO: 53) by One-step isothermal assembly.
- the obtained plasmid containing the expression cassette X15 was named pIBG-PGS.
- the promoter region of the gene PGK1 derived from Saccharomyces cerevisiae was prepared by PCR using pGK403 as a template and a primer pair PGK1p-F (SEQ ID NO: 50) and PGK1p-R2 (SEQ ID NO: 54). This fragment was ligated to the fragment amplified with the primer pair Sed1ss-F2 (SEQ ID NO: 55) and PRS-R3 (SEQ ID NO: 53) using the vector plasmid pIBG-SSS as a template by One-step isothermal assembly. The obtained plasmid containing the expression cassette X16 was named pIBG-PSS.
- the promoter region of the gene PGK1 derived from Saccharomyces cerevisiae was prepared by PCR using pGK403 as a template and a primer pair PGK1p-F (SEQ ID NO: 50) and PGK1p-R3 (SEQ ID NO: 56). This fragment was ligated to the fragment amplified by using the vector plasmid pIBG-SMS as a template and the primer pair MFA-F3 (SEQ ID NO: 57) and PRS-R3 (SEQ ID NO: 53) by One-step isothermal assembly.
- the obtained plasmid containing the expression cassette X17 was named pIBG-PMS.
- the promoter region of the cell surface localized protein gene CWP2 derived from Saccharomyces cerevisiae was subjected to PCR, using the Saccharomyces cerevisiae BY4741 genome as a template, primer pairs Cwp2p-F (SEQ ID NO: 58) and Cwp2p-R (SEQ ID NO: 59). It was prepared by amplifying using This fragment was ligated to the fragment amplified by using the vector plasmid pIBG-SSsec as a template and the primer pair Sed1ss-F3 (SEQ ID NO: 60) and PRS-R4 (SEQ ID NO: 61) by One-step isothermal assembly. The obtained plasmid containing the expression cassette X18 was named pIBG-CSsec.
- the promoter region and secretory signal sequence of the cell surface localized protein gene CWP2 derived from Saccharomyces cerevisiae were obtained by PCR using the Saccharomyces cerevisiae BY4741 genome as a template, primer pairs Cwp2p-F (SEQ ID NO: 58) and Cwp2ss-R ( It was prepared by amplification using SEQ ID NO: 62).
- This fragment was ligated to the fragment amplified by using the vector plasmid pIBG-SSsec as a template and the primer pair BGL1-F4 (SEQ ID NO: 63) and PRS-R4 (SEQ ID NO: 61) by One-step isothermal assembly.
- the obtained plasmid containing the expression cassette X19 was named pIBG-CCsec.
- the promoter region of the TDH1 gene derived from Pichia pastoris, the SpeI site, the MF ⁇ prepro leader sequence derived from Saccharomyces cerevisiae, the XhoI site, the green fluorescent protein (GFP) coding region, and the terminator region of the AOX1 gene derived from Pichia pastoris The placed fragments were gene-synthesized with the sites of restriction enzymes HindIII and BamHI at both ends.
- GFP a sea urchin mushroom green 1 (mUkG1) gene optimized for a Pichia pastoris codon was used. This fragment was treated with HindIII and BamHI and ligated to the similarly treated plasmid pUC19.
- the obtained plasmid was designated as pmUkG1_MF ⁇ .
- the G418 resistance gene sequence was amplified by PCR using the plasmid pPIC9K manufactured by Life Technology as a template and using the primer pair G418r-BamHI-F (SEQ ID NO: 64) and G418r-EcoRI-R (SEQ ID NO: 65). It was prepared by doing. This fragment was treated with BamHI and EcoRI and ligated to the similarly treated plasmid pmUkG1_MF ⁇ .
- the obtained plasmid containing the expression cassette X20 was named pGmUkG1_MF ⁇ .
- the secretory signal peptide sequence of Rhizopus oryzae-derived glucoamylase was obtained by PCR, using pIBG-SGS as a template, primer pair MFa-SpeI-F (SEQ ID NO: 66) and MFa-XhoI-R (SEQ ID NO: 67). Prepared by amplification. This fragment was treated with SpeI and XhoI, and similarly ligated to the plasmid pGmUkG1_MF ⁇ except for the MF ⁇ prepro leader sequence. The obtained plasmid containing the expression cassette X21 was named pGmUkG1_GA.
- Saccharomyces cerevisiae-derived SED1 secretion signal peptide sequence by PCR, the Saccharomyces cerevisiae BY4741 genome as a template, primer pairs Sed1ss-SpeI-F (SEQ ID NO: 68) and Sed1ss-XhoI-R (SEQ ID NO: 69) And prepared by amplification.
- This fragment was treated with SpeI and XhoI, and similarly ligated to the plasmid pGmUkG1_MF ⁇ except for the MF ⁇ prepro leader sequence.
- the obtained plasmid containing the expression cassette X22 was named pGmUkG1_SED1.
- Preparation Example 2 Preparation of various transformed yeasts
- the following plasmids described in Preparation Example 1 pIBG-SGS, pIBG-SSS, pIBG-SMS, pIBG-SHS, pIBG-SGsec, pIBG-SSsec, pIBG-SMsec, pIBG-SHsec, pIEG-SGS, pIEG-SSS, pIEG-SMS, pIBG-TGS, pIBG-TSS, pIBG-TMS, pIBG-PGS, pIBG-PSS, pIBG-PMS, pIBG-CSsec, and pIBG-CCsec
- yeast Saccharomyces cerevisiae BY4741 strain MAT ⁇ his3 leu2 met15 ura3 strain
- BY-BG-SGS BY-BG-SSS
- BY-BG-SMS BY-BG-SHS
- BY-BG-SGsec BY-BG-SSsec
- BY-BG-SSsec BY, respectively.
- -BG-SMsec BY-BG-SHsec
- BY-EG-SGS BY-EG-SSS
- BY-EG-SMS BY-BG-TGS
- BY-BG-TSS BY-
- BG-TMS strain BY-BG-PGS strain
- BY-BG-PSS strain BY-BG-PMS strain
- BY-BG-CSsec strain BY-BG-CCsec strain.
- plasmids (pGmUkG1_MFa, pGmUkG1_GA, and pGmUkG1_SED1) described in Preparation Example 1 were treated with BsiWI, respectively, and used for yeast Pichia pastoris CBS7435 strain (wild strain), and transformed by the lithium acetate method. These transformed strains are referred to as PP-GFP-MF ⁇ strain, PP-GFP-GA strain, and PP-GFP-SED1 strain, respectively.
- BGL activity activity amount per dry cell weight (U)
- U activity amount per dry cell weight
- U activity amount per liter of medium
- ⁇ -Glucosidase (BGL) activity was examined according to the following procedure.
- the culture solutions were collected every 24 hours from the start of the main culture, and the cells and the medium were separated by centrifugation at 1,000 g for 5 minutes.
- Measurement of the ⁇ -glucosidase activity of the bacterial cells was performed as follows: (1) Wash the cells twice with distilled water; (2) Reaction solution 500 ⁇ L (composition: 10 mM pNPG (p-nitrophenyl- ⁇ -D-glucopyranoside) 100 ⁇ L (final concentration 2 mM); 500 mM sodium citrate buffer (pH 5.0) 50 ⁇ L (final concentration 50 mM); distilled water 250 ⁇ L; and yeast suspension 100 ⁇ L) (final cell concentration 1-10 g wet cells / L)) and reaction at 500 rpm, 30 ° C.
- Reaction solution 500 ⁇ L composition: 10 mM pNPG (p-nitrophenyl- ⁇ -D-glucopyranoside) 100 ⁇ L (final concentration 2 mM); 500 mM sodium citrate buffer (pH 5.0) 50 ⁇ L (final concentration 50 mM); distilled water 250 ⁇ L; and yeast suspension 100 ⁇ L) (final cell concentration 1-10 g
- the reaction solution was prepared as described above except that 100 ⁇ L of the medium was added instead of the yeast suspension.
- Measurement of the endoglucanase activity of the bacterial cells was performed as follows: (1) Wash the cells twice with distilled water; (2) 2500 ⁇ L of reaction solution (composition: 1 tablet of cerazyme C tablet (manufactured by Megazyme); 250 ⁇ L of 500 mM sodium citrate buffer (pH 5.0) (final concentration 50 mM); 2000 ⁇ L of distilled water; and 250 ⁇ L of yeast suspension (final) Bacterial cell concentration 10g wet cell / L)) is prepared and allowed to stand at 38 ° C. for 4 hours; (3) After completion of the reaction, the mixture was centrifuged at 10,000 g for 5 minutes, and the absorbance ABS 590 at 590 nm of the supernatant was measured.
- the cells were transplanted into 500 ⁇ L of BMGY medium placed in a 96-well deep well plate, cultured at 30 ° C. and 1800 rpm for 18 hours (preculture), and then each preculture was freshly placed in a 96-well deep well plate. Transplanted into 500 ⁇ L of BMGY medium and cultured at 30 ° C. and 1800 rpm (main culture). 24 hours after the start of the main culture, the 96-well deep well plate was centrifuged at 3,000 rpm for 5 minutes to obtain respective culture supernatants.
- Example 1 Comparison of various secretion signals in ⁇ -glucosidase surface display
- various surface display transformed yeasts BY-BG-SGS, BY-BG-SSS, BY-BG-SMS obtained by introducing plasmids containing expression cassettes X1 to X4. Strain and BY-BG-SHS strain), the BGL activity of the bacterial cells was measured.
- FIG. 1 The result is shown in FIG.
- the horizontal axis in FIG. 1 indicates the culture time (“time (hour)”), and the vertical axis indicates ⁇ -glucosidase activity (activity per dry cell weight (“U / g dry cell weight”)).
- 1 are as follows: black circles, SED1 secretion signal (SED1); black diamond, Rhizopus oryzae-derived glucoamylase secretion signal (GA); black triangle, MF ⁇ prepro (MF ⁇ ); and black square, HKR1 Secretion signal (HKR1).
- BGL surface display transformed strains using the SED1 secretion signal are MF ⁇ prepro, which has been conventionally used for highly efficient expression of secreted proteins, and Rhizopus, which is often used for the expression of surface display proteins. Even when compared with a transformant using an oryzae-derived glucoamylase secretion signal, it was found that the surface BGL activity was considerably high.
- Example 2 Comparison of various secretion signals in ⁇ -glucosidase secretion
- various secretory transformed yeasts BY-BG-SGsec strain, BY-BG-SSsec strain, BY-BG-SMsec strain
- BY-BG-SHsec strain obtained by introducing plasmids containing expression cassettes of X5 to X8.
- BY-BG-SHsec strain obtained by introducing plasmids containing expression cassettes of X5 to X8.
- BY-BG-SHsec strain BGL activity in the medium was measured.
- FIG. 2 represents the culture time (“time (hour)”), and the vertical axis represents ⁇ -glucosidase activity (activity in the medium (“U / L”).
- SED1 secretion signal SED1
- GA glucoamylase secretion signal
- MF ⁇ MF ⁇ prepro
- HKR1 secretion signal HKR1 secretion signal
- BGL secretion transformed strains using SED1 secretion signal are MF ⁇ prepro, which has been conventionally used for highly efficient secretion protein expression, and Rhizopus oryzae, which is often used for expression of surface layer display proteins. Even when compared with the transformed strain using the derived glucoamylase secretion signal, it was found that the BGL activity was considerably high.
- Example 3 Comparison of various secretion signals in endoglucanase surface display
- various surface-displayed converted yeasts BY-EG-SGS, BY-EG-SSS, and BY-EG-SMS obtained by introducing plasmids containing expression cassettes X9 to X11. EG activity of the bacterial cells was measured.
- FIG. 3 indicates EG activity (absorbance measurement (“ABS590”).
- ABS590 absorbance measurement
- the bar graph in FIG. 3 shows, from left to right, lysopus oryzae-derived glucoamylase secretion signal (GA), MF ⁇ prepro (MF ⁇ ), and SED1 secretion).
- Signal (SED1) is represented.
- the surface-displayed transformant using the SED1 secretion signal is higher than the surface-displayed transformant using the secretion signal that has been commonly used conventionally. It was observed to show secretion efficiency.
- Example 4 Examination of combinations with various promoters
- various surface display converted yeasts BY-BG-TGS strain, BY-BG-TSS strain, BY-BG-TMS strain
- BY-BG-PGS strain, BY-BG-PSS strain, and BY-BG-PMS strain obtained by introducing plasmids containing expression cassettes X12 to X17.
- the BGL activity of the bacterial cells was measured.
- FIG. 4 The results are shown in FIG. 4 (upper graph: TDH3 promoter; and lower graph: PGK1 promoter).
- the horizontal axis of each graph in FIG. 4 represents the culture time (“time (hours)”), and the vertical axis represents ⁇ -glucosidase activity (activity per dry cell weight (“U / g dry cell weight”). 4 are as follows: black square, SED1 secretion signal (SED1); black rhombus, Rhizopus oryzae-derived glucoamylase secretion signal (GA); and black triangle, MF ⁇ prepro (MF ⁇ ) .
- the strain using the SED1 secretion signal has a high enzyme equivalent to or higher than the surface-layer-transformed strain using the secretion signal commonly used in the past. It was observed that the activity was shown stably. In particular, in the TDH3 promoter, the rate of increase in activity was remarkable as in the case of combining with the SED1 promoter shown in FIG.
- Example 5 Examination of combinations with various promoters
- various secretory transformed yeasts BY-BG-CSsec strain and BY-BG-CCsec strain obtained by introducing plasmids containing expression cassettes of X18 and X19 in the medium were used. BGL activity was measured.
- FIG. 5 This result is shown in FIG. 5, the horizontal axis indicates the culture time (“time (hour)”), and the vertical axis indicates ⁇ -glucosidase activity (activity in the medium (“U / L”). Symbols in FIG. The following are: black diamond, CWP2 promoter + SED1 secretion signal; and black square, CWP2 promoter + CWP2 secretion signal.
- the secretion transformant used in combination with the SED1 secretion signal is higher than the secretion transformant used in combination with the CWP2 secretion signal derived from the same gene. It was observed to show BGL activity.
- Example 6 Examination of secretory ability in heterologous yeast
- GFP-secreting transformants of yeast Pichia pastoris obtained by introducing plasmids containing expression cassettes of X20, X21 and X22 (PP-GFP-MF ⁇ strain, PP-GFP-GA strain, And the PP-GFP-SED1 strain)), the GFP fluorescence intensity in the medium was measured.
- FIG. 6 shows the relative value of the GFP fluorescence intensity in the medium of each strain when the GFP fluorescence intensity in the medium of the PP-GFP-MF ⁇ strain is 1.
- the bar graph of FIG. 6 represents, in order from the left: MF ⁇ prepro (MF ⁇ ), Rhizopus oryzae-derived glucoamylase secretion signal (GA), and SED1 secretion signal (SED1).
- the secretory transformant using the SED1 secretion signal is considerably more than the secretory transformant using the secretory signal that has been conventionally used. It was observed to show high GFP fluorescence intensity. From these results, it was shown that the SED1 secretion signal can improve the secreted amount of the protein even when the yeast species other than Saccharomyces cerevisiae are used as the host.
- various proteins such as enzymes can be efficiently secreted outside the cell or presented on the cell surface. This is very useful for increasing the efficiency of production of substances using yeast, reducing the cost, and promoting their spread. More specific fields of application include the production of proteins such as antibodies and enzymes, the efficiency of production of chemicals from cellulosic biomass using yeast that presents a cellulolytic enzyme on the cell surface, and cost reduction.
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Abstract
Description
(i)プロモーターDNA、
(ii)以下:
(a)配列番号2で表されるアミノ酸配列からなるペプチド、
(b)配列番号2で表されるアミノ酸配列と少なくとも70%の配列同一性を有するアミノ酸配列を有し、分泌シグナル活性を有するペプチド、
(c)配列番号2で表されるアミノ酸配列に対して1または数個のアミノ酸残基を置換、欠失または付加して得られるアミノ酸配列を有し、分泌シグナル活性を有するペプチド、
(d)配列番号1で表される塩基配列と少なくとも70%の配列同一性を有する塩基配列によってコードされ、分泌シグナル活性を有するペプチド、および
(e)配列番号1で表される塩基配列からなるDNAの相補鎖とハイブリダイズする塩基配列によってコードされ、分泌シグナル活性を有するペプチド、
からなる群より選択されるいずれかのペプチドをコードするDNA;ならびに
(iii)目的タンパク質をコードするDNA、または該目的タンパク質をコードするDNAを挿入するためのクローニング部位。
プロモーターDNA;(a)配列番号2で表されるアミノ酸配列からなるペプチド、(b)配列番号2で表されるアミノ酸配列と少なくとも70%の配列同一性を有するアミノ酸配列を有し、分泌シグナル活性を有するペプチド、(c)配列番号2で表されるアミノ酸配列に対して1または数個のアミノ酸残基を置換、欠失または付加して得られるアミノ酸配列を有し、分泌シグナル活性を有するペプチド、(d)配列番号1で表される塩基配列と少なくとも70%の配列同一性を有する塩基配列によってコードされ、分泌シグナル活性を有するペプチド、および(e)配列番号1で表される塩基配列からなるDNAの相補鎖とハイブリダイズする塩基配列によってコードされ、分泌シグナル活性を有するペプチドからなる群より選択されるいずれかのペプチドをコードするDNA;ならびに目的タンパク質をコードするDNAを含む発現カセットを酵母に導入し、形質転換酵母を得る工程を含む。
(i)プロモーターDNA、
(ii)以下:
(a)配列番号2で表されるアミノ酸配列からなるペプチド;
(b)配列番号2で表されるアミノ酸配列と少なくとも70%の配列同一性を有するアミノ酸配列を有し、分泌シグナル活性を有するペプチド、
(c)配列番号2で表されるアミノ酸配列に対して1または数個のアミノ酸残基を置換、欠失または付加して得られるアミノ酸配列を有し、分泌シグナル活性を有するペプチド、
(d)配列番号1で表される塩基配列と少なくとも70%の配列同一性を有する塩基配列によってコードされ、分泌シグナル活性を有するペプチド、および
(e)配列番号1で表される塩基配列からなるDNAの相補鎖とハイブリダイズする塩基配列によってコードされ、分泌シグナル活性を有するペプチド、
からなる群より選択されるいずれかのペプチドをコードするDNA;
(iii)目的タンパク質をコードするDNA、または該目的タンパク質をコードするDNAを挿入するためのクローニング部位;ならびに、
(iv)アンカードメインをコードするDNA。
プロモーターDNA;(a)配列番号2で表されるアミノ酸配列からなるペプチド、(b)配列番号2で表されるアミノ酸配列と少なくとも70%の配列同一性を有するアミノ酸配列を有し、分泌シグナル活性を有するペプチド、(c)配列番号2で表されるアミノ酸配列に対して1または数個のアミノ酸残基を置換、欠失または付加して得られるアミノ酸配列を有し、分泌シグナル活性を有するペプチド、(d)配列番号1で表される塩基配列と少なくとも70%の配列同一性を有する塩基配列によってコードされ、分泌シグナル活性を有するペプチド、および(e)配列番号1で表される塩基配列からなるDNAの相補鎖とハイブリダイズする塩基配列によってコードされ、分泌シグナル活性を有するペプチドからなる群より選択されるいずれかのペプチドをコードするDNA;目的タンパク質をコードするDNA;ならびにアンカードメインをコードするDNAを含む発現カセットを酵母に導入し、形質転換酵母を得る工程、
を含む。
分泌シグナルペプチドは、細胞膜、細胞壁に局在する、または細胞膜外に分泌されるタンパク質のN末端に通常結合しているペプチドである。分泌シグナルペプチドは、通常、分泌性タンパク質が細胞内から細胞膜を通過して細胞外へ分泌される過程でシグナルペプチダーゼにより分解されることにより除去される。例えば、分泌シグナルペプチドは、当該分泌シグナルペプチドを本来有するタンパク質(分泌性タンパク質)または異種タンパク質のN末端に、これらのタンパク質が発現されるべき細胞内で連結されることで、その分泌性タンパク質または異種タンパク質を細胞外に分泌させるように機能する。
(a)配列番号2で表されるアミノ酸配列からなるペプチド;
(b)配列番号2で表されるアミノ酸配列と少なくとも70%の配列同一性を有するアミノ酸配列を有し、分泌シグナル活性を有するペプチド;
(c)配列番号2で表されるアミノ酸配列に対して1または数個のアミノ酸残基を置換、欠失または付加して得られるアミノ酸配列を有し、分泌シグナル活性を有するペプチド;
(d)配列番号1で表される塩基配列と少なくとも70%の配列同一性を有する塩基配列によってコードされ、分泌シグナル活性を有するペプチド;および
(e)配列番号1で表される塩基配列からなるDNAの相補鎖とハイブリダイズする塩基配列によってコードされ、分泌シグナル活性を有するペプチド。
表層提示カセットは、アンカードメインをコードするDNAを含む。
プロモーターDNAは、プロモーター活性を有していればよく、「プロモーター活性」とは、プロモーター領域に転写因子が結合し、転写を惹起する活性をいう。プロモーターDNAは、所望のプロモーター領域を保有する菌体、ファージなどから、制限酵素を用いて切り出し得る。必要に応じて制限酵素認識部位またはクローニングベクターとの重複部位を設けたプライマーを用い、PCRで所望のプロモーター領域を増幅することによりプロモーター領域のDNA断片を得ることができる。また、既に判明しているプロモーター領域の塩基配列情報をもとにして、所望のプロモーターDNAを化学合成してもよい。
目的タンパク質の種類、もしくはその起源は特に限定されない。目的タンパク質の種類として、例えば、酵素、抗体、リガンド、蛍光タンパク質などが挙げられる。酵素としては、例えば、セルロース分解酵素、デンプン分解酵素、グリコーゲン分解酵素、キシラン分解酵素、キチン分解酵素、脂質分解酵素などが挙げられ、より具体的には、例えば、エンドグルカナーゼ、セロビオヒドロラーゼ、およびβ-グルコシダーゼ、アミラーゼ(例えば、グルコアミラーゼおよびα-アミラーゼ)、リパーゼなどが挙げられる。
分泌カセットまたは表層提示カセットを含むポリヌクレオチドは、さらにターミネーターDNAを含むことができる。
本明細書において、「発現カセット」とは、目的タンパク質が発現し得るように、該目的タンパク質をコードするDNAと、その発現を調節するための種々の調節エレメントとが、宿主の微生物または細胞中で機能し得る状態で連結されているDNA配列またはポリヌクレオチドをいう。ここで「機能し得る状態で連結されている」とは、目的タンパク質をコードするDNAが、プロモーターの制御下で、そして場合により他の調節エレメントの制御下で、発現されるように、発現カセットまたは発現ベクターに含まれる各構成要素が連結されていることを意味する。各構成要素は、目的タンパク質が発現し得る限りにおいて、それらの要素間にリンカーなどの配列をさらに含んで連結されていてもよい。
本発明は、上記発現カセットを含む発現ベクターを提供する。本発明の分泌型の発現ベクターは、上記分泌型の発現カセットを含み、そして本発明の表層提示型の発現ベクターは、上記表層提示型の発現カセットを含む。本明細書において「発現ベクター」とは、目的タンパク質をコードするDNAの発現のためのユニット(発現カセット)が挿入されたベクターをいい、目的タンパク質をコードするDNAが挿入されたものを含む。発現ベクターは、プラスミドベクターであってもよく、あるいは人工染色体であってもよい。酵母を宿主とする場合、ベクターの調製が容易であり、また酵母細胞の形質転換が容易である点で、プラスミドの形態が好ましい。DNAの取得の簡易化の点からは、酵母と大腸菌とのシャトルベクターであることが好ましい。必要に応じて、ベクターは、調節配列(オペレーター、エンハンサーなど)を含み得る。このようなベクターは、例えば、酵母の2μmプラスミドの複製開始点(Ori)とColE1の複製開始点とを有しており、酵母選択マーカー(以下に説明)および大腸菌の選択マーカー(薬剤耐性遺伝子など)を有する。
宿主として用いる酵母は、子のう菌酵母(Ascomycetous yeast)に属するものであればよく、特に限定されない。例えば、サッカロマイセス属(Saccharomyces)、クルイウェロマイセス属(Kluyveromyces)、カンジダ属(Candida)、ピキア属(Pichia)、スキゾサッカロマイセス属(Schizosaccharomyces)、ハンセヌラ属(Hancenula)、クロッケラ属(Kloeckera)、シュワニオマイセス属(Schwanniomyces)、コマガタエラ属(Komagataella)およびヤロウィア属(Yarrowia)などの酵母が挙げられる。
下記発現カセットX1~X19をそれぞれ含むプラスミドを調製した:
X1:SED1プロモーター+グルコアミラーゼ分泌シグナル+BGL1+SED1アンカードメイン
X2:SED1プロモーター+SED1分泌シグナル+BGL1+SED1アンカードメイン
X3:SED1プロモーター+MFα prepro+BGL1+SED1アンカードメイン
X4:SED1プロモーター+HKR1分泌シグナル+BGL1+SED1アンカードメイン
X5:SED1プロモーター+グルコアミラーゼ分泌シグナル+BGL1
X6:SED1プロモーター+SED1分泌シグナル+BGL1
X7:SED1プロモーター+MFα prepro+BGL1
X8:SED1プロモーター+HKR1分泌シグナル+BGL1
X9:SED1プロモーター+グルコアミラーゼ分泌シグナル+EGII+SED1アンカードメイン
X10:SED1プロモーター+SED1分泌シグナル+EGII+SED1アンカードメイン
X11:SED1プロモーター+MFα prepro+EGII+SED1アンカードメイン
X12:TDH3プロモーター+グルコアミラーゼ分泌シグナル+BGL1+SED1アンカードメイン
X13:TDH3プロモーター+SED1分泌シグナル+BGL1+SED1アンカードメイン
X14:TDH3プロモーター+MFα prepro+BGL1+SED1アンカードメイン
X15:PGK1プロモーター+グルコアミラーゼ分泌シグナル+BGL1+SED1アンカードメイン
X16:PGK1プロモーター+SED1分泌シグナル+BGL1+SED1アンカードメイン
X17:PGK1プロモーター+MFα prepro+BGL1+SED1アンカードメイン
X18:CWP2プロモーター+SED1分泌シグナル+BGL1
X19:CWP2プロモーター+CWP2分泌シグナル+BGL1
X20:ピキア・パストリス由来TDH1プロモーター+MFα prepro+GFP
X21:ピキア・パストリス由来TDH1プロモーター+グルコアミラーゼ分泌シグナル+GFP
X22:ピキア・パストリス由来TDH1プロモーター+SED1分泌シグナル+GFP
配列番号1:サッカロマイセス・セレビシエ由来のSED1の分泌シグナルペプチドをコードするDNAの塩基配列
配列番号2:サッカロマイセス・セレビシエ由来のSED1の分泌シグナルペプチドのアミノ酸配列
配列番号3:サッカロマイセス・セレビシエ由来のSED1のプロモーターの塩基配列
配列番号4:本発明の実施例においてSED1アンカードメインとして用いた、サッカロマイセス・セレビシエ由来のSED1アンカータンパク質のコーディング領域から開始コドンを除いた領域のDNAの塩基配列
配列番号5:本発明の実施例においてSED1アンカードメインとして用いた、サッカロマイセス・セレビシエ由来のSED1アンカータンパク質(但し開始メチオニンを含まない)のアミノ酸配列
配列番号6:リゾプス・オリゼ(Rhizopus oryzae)由来のグルコアミラーゼ分泌シグナルペプチドをコードするDNAの塩基配列
配列番号7:リゾプス・オリゼ由来のグルコアミラーゼ分泌シグナルペプチドのアミノ酸配列
配列番号8:サッカロマイセス・セレビシエ由来のMFα preproをコードするDNAの塩基配列
配列番号9:サッカロマイセス・セレビシエ由来のMFα preproのアミノ酸配列
配列番号10:サッカロマイセス・セレビシエ由来の分泌タンパク質HKR1の分泌シグナルペプチドをコードするDNAの塩基配列
配列番号11:サッカロマイセス・セレビシエ由来の分泌タンパク質HKR1の分泌シグナルペプチドのアミノ酸配列
配列番号12:サッカロマイセス・セレビシエ由来のTDH3プロモーターの塩基配列
配列番号13:サッカロマイセス・セレビシエ由来のPGK1プロモーターの塩基配列
配列番号14:アスペルギルス・アクレアタス由来β-グルコシダーゼ1(BGL1)をコードするDNAの塩基配列
配列番号15:アスペルギルス・アクレアタス由来β-グルコシダーゼ1(BGL1)のアミノ酸配列
配列番号16:トリコデルマ・リーセイ由来エンドグルカナーゼII(EGII)をコードするDNAの塩基配列
配列番号17:トリコデルマ・リーセイ由来エンドグルカナーゼII(EGII)のアミノ酸配列
配列番号18:サッカロマイセス・セレビシエ由来のCWP2プロモーターの塩基配列
配列番号19:サッカロマイセス・セレビシエ由来のCWP2分泌シグナルペプチドをコードするDNAの塩基配列
配列番号20:サッカロマイセス・セレビシエ由来のCWP2分泌シグナルペプチドのアミノ酸配列
配列番号21:ピキア・パストリス由来のTDH1プロモーターの塩基配列
配列番号22:ピキア・パストリスのコドンに最適化したウミキノコグリーン1(mUkG1)をコードするDNAの塩基配列
配列番号23:ウミキノコグリーン1(mUkG1)のアミノ酸配列
調製例1に記載の以下のプラスミド(pIBG-SGS、pIBG-SSS、pIBG-SMS、pIBG-SHS、pIBG-SGsec、pIBG-SSsec、pIBG-SMsec、pIBG-SHsec、pIEG-SGS、pIEG-SSS、pIEG-SMS、pIBG-TGS、pIBG-TSS、pIBG-TMS、pIBG-PGS、pIBG-PSS、pIBG-PMS、pIBG-CSsec、およびpIBG-CCsec)をNdeIで処理し、それぞれ酵母サッカロマイセス・セレビシエBY4741株(MATα his3 leu2 met15 ura3株)に供し、酢酸リチウム法により形質転換した。これらの形質転換株をそれぞれBY-BG-SGS株、BY-BG-SSS株、BY-BG-SMS株、BY-BG-SHS株、BY-BG-SGsec株、BY-BG-SSsec株、BY-BG-SMsec株、BY-BG-SHsec株、BY-EG-SGS株、BY-EG-SSS株、BY-EG-SMS株、BY-BG-TGS株、BY-BG-TSS株、BY-BG-TMS株、BY-BG-PGS株、BY-BG-PSS株、BY-BG-PMS株、BY-BG-CSsec株、およびBY-BG-CCsec株と称する。
表層提示株については菌体のBGL活性(乾燥菌体重量当たりの活性量(U))を、そして分泌株については培地中のBGL活性(培地1L当たりの活性量(U))を測定した。β-グルコシダーゼ(BGL)活性の検討を以下の手順に従って行った。
(1)菌体を蒸留水で2回洗浄;
(2)反応液500μL(組成:10mM pNPG(p-ニトロフェニル-β-D-グルコピラノシド)100μL(最終濃度2mM);500mM クエン酸ナトリウム緩衝液(pH5.0) 50μL(最終濃度50mM);蒸留水250μL;および酵母懸濁液100μL)(最終菌体濃度1~10g湿潤菌体/L))を調製し、500rpm、30℃にて10分間反応;
(3)反応終了後、3M Na2CO3 500μLを加え反応を停止;そして
(4)10,000gで5分間遠心後、上清の400nmにおける吸光度ABS400を測定。1分間で1μmolのpNP(p-ニトロフェノール)を遊離する酵素量を1Uとする。
表層提示株について、菌体のEG活性(乾燥菌体重量当たりの活性量(U))を以下の手順に従って行った。
(1)菌体を蒸留水で2回洗浄;
(2)反応液2500μL(組成:セラザイムCタブレット(Megazyme社製)1錠;500mM クエン酸ナトリウム緩衝液(pH5.0) 250μL(最終濃度50mM);蒸留水2000μL;および酵母懸濁液250μL(最終菌体濃度10g湿潤菌体/L))を調製し、静置、38℃にて4時間反応;
(3)反応終了後、10,000gで5分間遠心後、上清の590nmの吸光度ABS590を測定。
キア・パストリスのGFP分泌株については培地中のGFP蛍光強度を以下の手順に従って行った。
本実施例では、X1~X4のそれぞれの発現カセットを含むプラスミドを導入して得られた各種の表層提示形質転換酵母(BY-BG-SGS株、BY-BG-SSS株、BY-BG-SMS株、およびBY-BG-SHS株)について、菌体のBGL活性を測定した。
本実施例では、X5~X8のそれぞれの発現カセットを含むプラスミドを導入して得られた各種の分泌形質転換酵母(BY-BG-SGsec株、BY-BG-SSsec株、BY-BG-SMsec株、およびBY-BG-SHsec株)について、培地中のBGL活性を測定した。
本実施例では、X9~X11のそれぞれの発現カセットを含むプラスミドを導入して得られた各種の表層提示転換酵母(BY-EG-SGS株、BY-EG-SSS株、およびBY-EG-SMS株)について、菌体のEG活性を測定した。
本実施例では、X12~X17のそれぞれの発現カセットを含むプラスミドを導入して得られた各種の表層提示転換酵母(BY-BG-TGS株、BY-BG-TSS株、BY-BG-TMS株、BY-BG-PGS株、BY-BG-PSS株、およびBY-BG-PMS株)について、菌体のBGL活性を測定した。
本実施例では、X18およびX19のそれぞれの発現カセットを含むプラスミドを導入して得られた各種の分泌形質転換酵母(BY-BG-CSsec株、およびBY-BG-CCsec株)について、培地中のBGL活性を測定した。
本実施例では、X20、X21およびX22のそれぞれの発現カセットを含むプラスミドを導入して得られた酵母ピキア・パストリスのGFP分泌形質転換体(PP-GFP-MFα株、PP-GFP-GA株、およびPP-GFP-SED1株と称する。)について、培地中のGFP蛍光強度を測定した。
Claims (12)
- 以下の(i)、(ii)および(iii)を含む、発現ベクター:
(i)プロモーターDNA、
(ii)以下:
(a)配列番号2で表されるアミノ酸配列からなるペプチド、
(b)配列番号2で表されるアミノ酸配列と少なくとも70%の配列同一性を有するアミノ酸配列を有し、分泌シグナル活性を有するペプチド、
(c)配列番号2で表されるアミノ酸配列に対して1または数個のアミノ酸残基を置換、欠失または付加して得られるアミノ酸配列を有し、分泌シグナル活性を有するペプチド、
(d)配列番号1で表される塩基配列と少なくとも70%の配列同一性を有する塩基配列によってコードされ、分泌シグナル活性を有するペプチド、および
(e)配列番号1で表される塩基配列からなるDNAの相補鎖とハイブリダイズする塩基配列によってコードされ、分泌シグナル活性を有するペプチド、
からなる群より選択されるいずれかのペプチドをコードするDNA;ならびに
(iii)目的タンパク質をコードするDNA、または該目的タンパク質をコードするDNAを挿入するためのクローニング部位。 - 前記プロモーターがSED1プロモーターである、請求項1に記載の発現ベクター。
- 前記(iii)が目的タンパク質をコードするDNAである、請求項1または2に記載の発現ベクター。
- 請求項3に記載の発現ベクターが導入された形質転換酵母。
- タンパク質を分泌生産する酵母の作製方法であって、
プロモーターDNA;(a)配列番号2で表されるアミノ酸配列からなるペプチド、(b)配列番号2で表されるアミノ酸配列と少なくとも70%の配列同一性を有するアミノ酸配列を有し、分泌シグナル活性を有するペプチド、(c)配列番号2で表されるアミノ酸配列に対して1または数個のアミノ酸残基を置換、欠失または付加して得られるアミノ酸配列を有し、分泌シグナル活性を有するペプチド、(d)配列番号1で表される塩基配列と少なくとも70%の配列同一性を有する塩基配列によってコードされ、分泌シグナル活性を有するペプチド、および(e)配列番号1で表される塩基配列からなるDNAの相補鎖とハイブリダイズする塩基配列によってコードされ、分泌シグナル活性を有するペプチドからなる群より選択されるいずれかのペプチドをコードするDNA;ならびに目的タンパク質をコードするDNAを含む発現カセットを酵母に導入し、形質転換酵母を得る工程、
を含む、方法。 - 酵母においてタンパク質を分泌生産する方法であって、
請求項4に記載の形質転換酵母または請求項5に記載の方法により作製された酵母を培養する工程
を含む、方法。 - 以下の(i)、(ii)、(iii)および(iv)を含む、発現ベクター:
(i)プロモーターDNA、
(ii)以下:
(a)配列番号2で表されるアミノ酸配列からなるペプチド;
(b)配列番号2で表されるアミノ酸配列と少なくとも70%の配列同一性を有するアミノ酸配列を有し、分泌シグナル活性を有するペプチド、
(c)配列番号2で表されるアミノ酸配列に対して1または数個のアミノ酸残基を置換、欠失または付加して得られるアミノ酸配列を有し、分泌シグナル活性を有するペプチド、
(d)配列番号1で表される塩基配列と少なくとも70%の配列同一性を有する塩基配列によってコードされ、分泌シグナル活性を有するペプチド、および
(e)配列番号1で表される塩基配列からなるDNAの相補鎖とハイブリダイズする塩基配列によってコードされ、分泌シグナル活性を有するペプチド、
からなる群より選択されるいずれかのペプチドをコードするDNA;
(iii)目的タンパク質をコードするDNA、または該目的タンパク質をコードするDNAを挿入するためのクローニング部位;ならびに、
(iv)アンカードメインをコードするDNA。 - 前記プロモーターがSED1プロモーターである、請求項7に記載の発現ベクター。
- 前記アンカードメインがSED1アンカードメインである、請求項7または8に記載の発現ベクター。
- 前記(iii)が目的タンパク質をコードするDNAである、請求項7から9のいずれかに記載の発現ベクター。
- 請求項10に記載の発現ベクターが導入された形質転換酵母。
- タンパク質を表層提示する酵母の作製方法であって、
プロモーターDNA;(a)配列番号2で表されるアミノ酸配列からなるペプチド、(b)配列番号2で表されるアミノ酸配列と少なくとも70%の配列同一性を有するアミノ酸配列を有し、分泌シグナル活性を有するペプチド、(c)配列番号2で表されるアミノ酸配列に対して1または数個のアミノ酸残基を置換、欠失または付加して得られるアミノ酸配列を有し、分泌シグナル活性を有するペプチド、(d)配列番号1で表される塩基配列と少なくとも70%の配列同一性を有する塩基配列によってコードされ、分泌シグナル活性を有するペプチド、および(e)配列番号1で表される塩基配列からなるDNAの相補鎖とハイブリダイズする塩基配列によってコードされ、分泌シグナル活性を有するペプチドからなる群より選択されるいずれかのペプチドをコードするDNA;目的タンパク質をコードするDNA;ならびにアンカードメインをコードするDNAを含む発現カセットを酵母に導入し、形質転換酵母を得る工程、
を含む、方法。
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2019104699A (ja) * | 2017-12-11 | 2019-06-27 | 国立大学法人神戸大学 | 二重特異性抗体 |
CN110637085A (zh) * | 2017-03-13 | 2019-12-31 | 拉勒曼德匈牙利流动性管理有限责任公司 | 表达细胞相连的异源蛋白的重组酵母宿主细胞 |
JP2020510442A (ja) * | 2017-03-13 | 2020-04-09 | ラレマンド ハンガリー リクィディティー マネジメント エルエルシーLallemand Hungary Liquidity Management Llc | 細胞結合型異種タンパク質を発現する組換え酵母宿主細胞 |
JP2021090384A (ja) * | 2019-12-10 | 2021-06-17 | 国立大学法人神戸大学 | セルラーゼを細胞表層発現する形質転換酵母 |
US11306300B2 (en) | 2017-05-11 | 2022-04-19 | Kansai Chemical Engineering Co., Ltd. | Microorganism capable of displaying α-galactosidase on surface layer thereof, and use thereof |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11259923B2 (en) | 2013-03-14 | 2022-03-01 | Jc Medical, Inc. | Methods and devices for delivery of a prosthetic valve |
US11406497B2 (en) | 2013-03-14 | 2022-08-09 | Jc Medical, Inc. | Heart valve prosthesis |
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BR112021019076A2 (pt) * | 2019-03-25 | 2021-11-30 | Alteogen Inc | Composição farmacêutica e formulação para injeção subcutânea compreendendo uma variante de hialuronidase ph20 humana |
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2005538711A (ja) * | 2002-09-13 | 2005-12-22 | コリア リサーチ インスティチュート オブ バイオサイエンス アンド バイオテクノロジー | 酵母表面提示ベクターを利用して改良型酵素活性を有するリパーゼをスクリーニングする方法及びそのリパーゼ |
JP2007167062A (ja) * | 2005-11-24 | 2007-07-05 | National Institute Of Advanced Industrial & Technology | 高効率分泌シグナルペプチド及びそれらを利用したタンパク質発現系 |
JP2009183249A (ja) * | 2008-02-08 | 2009-08-20 | Gekkeikan Sake Co Ltd | バイオエタノールの製造方法 |
KR20120140577A (ko) * | 2011-06-21 | 2012-12-31 | 한국생명공학연구원 | 외래단백질 분비생산용 인공 단백질 분비융합인자, 이를 사용한 외래단백질의 생산방법 및 인공 단백질 분비융합인자의 스크리닝 방법 |
-
2015
- 2015-07-30 US US15/328,766 patent/US20170218382A1/en not_active Abandoned
- 2015-07-30 JP JP2016538422A patent/JP6537076B2/ja active Active
- 2015-07-30 WO PCT/JP2015/071608 patent/WO2016017736A1/ja active Application Filing
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2005538711A (ja) * | 2002-09-13 | 2005-12-22 | コリア リサーチ インスティチュート オブ バイオサイエンス アンド バイオテクノロジー | 酵母表面提示ベクターを利用して改良型酵素活性を有するリパーゼをスクリーニングする方法及びそのリパーゼ |
JP2007167062A (ja) * | 2005-11-24 | 2007-07-05 | National Institute Of Advanced Industrial & Technology | 高効率分泌シグナルペプチド及びそれらを利用したタンパク質発現系 |
JP2009183249A (ja) * | 2008-02-08 | 2009-08-20 | Gekkeikan Sake Co Ltd | バイオエタノールの製造方法 |
KR20120140577A (ko) * | 2011-06-21 | 2012-12-31 | 한국생명공학연구원 | 외래단백질 분비생산용 인공 단백질 분비융합인자, 이를 사용한 외래단백질의 생산방법 및 인공 단백질 분비융합인자의 스크리닝 방법 |
Non-Patent Citations (2)
Title |
---|
DATABASE GenBank [o] 30 June 2006 (2006-06-30), "Definition: S.cerevisiae SED1 gene", Database accession no. X66838 * |
KENTARO INOKUMA ET AL.: "Development of the novel secretion signal sequence for highly- efficient cell surface display and secretory production of proteins by yeasts", DAI 66 KAI ABSTRACTS OF THE ANNUAL MEETING OF THE SOCIETY FOR BIOTECHNOLOGY, 5 August 2014 (2014-08-05), Japan, pages 51, 1P-134 * |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110637085A (zh) * | 2017-03-13 | 2019-12-31 | 拉勒曼德匈牙利流动性管理有限责任公司 | 表达细胞相连的异源蛋白的重组酵母宿主细胞 |
JP2020510442A (ja) * | 2017-03-13 | 2020-04-09 | ラレマンド ハンガリー リクィディティー マネジメント エルエルシーLallemand Hungary Liquidity Management Llc | 細胞結合型異種タンパク質を発現する組換え酵母宿主細胞 |
JP7194112B2 (ja) | 2017-03-13 | 2022-12-21 | ラレマンド ハンガリー リクィディティー マネジメント エルエルシー | 細胞結合型異種タンパク質を発現する組換え酵母宿主細胞 |
US11306300B2 (en) | 2017-05-11 | 2022-04-19 | Kansai Chemical Engineering Co., Ltd. | Microorganism capable of displaying α-galactosidase on surface layer thereof, and use thereof |
JP2019104699A (ja) * | 2017-12-11 | 2019-06-27 | 国立大学法人神戸大学 | 二重特異性抗体 |
JP7072792B2 (ja) | 2017-12-11 | 2022-05-23 | 国立大学法人神戸大学 | 二重特異性抗体 |
JP2021090384A (ja) * | 2019-12-10 | 2021-06-17 | 国立大学法人神戸大学 | セルラーゼを細胞表層発現する形質転換酵母 |
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