WO2021187560A1 - Procédé de préparation de cellules génétiquement modifiées ayant une expression génique endogène améliorée - Google Patents

Procédé de préparation de cellules génétiquement modifiées ayant une expression génique endogène améliorée Download PDF

Info

Publication number
WO2021187560A1
WO2021187560A1 PCT/JP2021/011093 JP2021011093W WO2021187560A1 WO 2021187560 A1 WO2021187560 A1 WO 2021187560A1 JP 2021011093 W JP2021011093 W JP 2021011093W WO 2021187560 A1 WO2021187560 A1 WO 2021187560A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
base sequence
sequence represented
same
nucleic acid
Prior art date
Application number
PCT/JP2021/011093
Other languages
English (en)
Japanese (ja)
Inventor
洋一郎 伊藤
石井　純
近藤　昭彦
Original Assignee
国立大学法人神戸大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人神戸大学 filed Critical 国立大学法人神戸大学
Priority to JP2022508434A priority Critical patent/JPWO2021187560A1/ja
Publication of WO2021187560A1 publication Critical patent/WO2021187560A1/fr

Links

Images

Classifications

    • 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
    • C12N15/67—General methods for enhancing the expression
    • 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
    • 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 is a method for producing a recombinant cell in which the expression of an endogenous gene is enhanced, the recombinant cell produced by the method, and the production of a target protein using an endogenous gene overexpressing cell library containing the cell.
  • the present invention relates to a method for screening an endogenous gene that enhances.
  • the gene recombination method is widely used for the production of industrially useful biomaterials such as antibodies, enzymes, and cytokines for medical and diagnostic purposes.
  • Hosts for producing the target protein by the gene recombination method include animals such as chickens, animal cells such as CHO, insects such as silk moth, insect cells such as sf9, and microorganisms such as yeast, Escherichia coli, and actinomycetes. It is used.
  • yeast can be cultivated on a large scale and at high density in an inexpensive medium, so that the target protein can be produced at low cost, and if a signal peptide or the like is used, the target protein is secreted into the culture solution.
  • Komagataella pastoris a type of yeast, is a methanol-utilizing (Mut + ) yeast that has excellent protein expression capacity and can utilize an inexpensive carbon source that is advantageous for industrial production.
  • Non-Patent Document 1 reports a method for producing a heterologous protein such as green fluorescent protein, human serum albumin, hepatitis B virus surface antigen, human insulin, and single-chain antibody using Komagataera pastris. ing.
  • a heterologous protein such as green fluorescent protein, human serum albumin, hepatitis B virus surface antigen, human insulin, and single-chain antibody using Komagataera pastris. ing.
  • When heterologous proteins are produced in yeast in order to improve their productivity, addition of signal sequences, utilization of strong promoters, codon modification, co-expression of chaperon genes, co-expression of transcription factor genes, proteases derived from host yeast Various attempts have been made such as gene inactivation and examination of culture conditions.
  • the present invention is a method for producing a recombinant cell in which the expression of an endogenous gene is enhanced, the recombinant cell produced by the method, and the production of a target protein using an endogenous gene overexpressing cell library containing the cell. It is an object of the present invention to provide a screening method for an endogenous gene that enhances the protein.
  • a method for producing a recombinant cell in which the expression of an endogenous gene is enhanced which comprises the following steps.
  • (1) A step of preparing a linear nucleic acid by cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme, wherein the nucleic acid fragment is a highly expressive promoter and a partial sequence starting from the start codon of the endogenous gene.
  • a step comprising a partial sequence into which the restriction enzyme recognition site has been inserted and a base sequence in which stop codons are sequentially linked.
  • the endogenous gene is at least one endogenous gene selected from the group consisting of the following endogenous genes (1) to (32). the method of. (1) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 46. (2) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 47. (3) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 48. (4) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 49.
  • nucleic acid fragment is at least one nucleic acid fragment selected from the group consisting of the following nucleic acid fragments (i) to (xxxii).
  • nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 238 (I) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 238, (Ii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 239, (Iii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 240, (Iv) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 241.
  • nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 252 (Xvi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 253, (Xvii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 254, (Xviii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 255, (Xix) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 256, (Xx) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 257.
  • (Xxi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 258, (Xxii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 259, (Xxiii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 260, (Xxiv) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 261.
  • a gene set containing an endogenous gene in which a highly expressive promoter is operably linked in which a plasmid containing a nucleic acid fragment is homologously recombined with a linear nucleic acid in which a nucleic acid fragment is cleaved with a restriction enzyme.
  • the nucleic acid fragment was a highly expressive promoter, a partial sequence starting from the start codon of the endogenous gene, and the partial sequence into which the restriction enzyme recognition site was inserted and the end codon were sequentially linked.
  • a recombinant cell containing a base sequence.
  • the endogenous gene is at least one endogenous gene selected from the group consisting of the following endogenous genes (1) to (32). Genetically modified cells.
  • nucleic acid fragment is at least one nucleic acid fragment selected from the group consisting of the following nucleic acid fragments (i) to (xxxii).
  • nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 238 (I) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 238, (Ii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 239, (Iii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 240, (Iv) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 241.
  • nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 252 (Xvi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 253, (Xvii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 254, (Xviii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 255, (Xix) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 256, (Xx) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 257.
  • (Xxi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 258, (Xxii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 259, (Xxiii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 260, (Xxiv) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 261.
  • [13] The genetically modified cell according to any one of [7] to [12], which comprises a base sequence encoding a target protein in its genome.
  • a method for producing a target protein which comprises the step of culturing the recombinant cells according to [13] or [14].
  • a method for screening an endogenous gene that enhances the production of a target protein which comprises the following steps.
  • a method for screening an endogenous gene that enhances the production of a target protein which comprises the following steps.
  • each endogenous gene By providing a method for enhancing the expression of each endogenous gene, it has become possible to easily construct a transgenic cell library in which the expression of each endogenous gene is enhanced. In addition, the use of the library has made it possible to screen for endogenous genes that increase the production of the target protein.
  • the present invention is a method for producing a recombinant cell with enhanced expression of an endogenous gene (hereinafter, the method for producing a recombinant cell of the present invention). I will provide a.
  • the recombinant cell refers to a cell in which a linear nucleic acid described later is introduced and the expression of an endogenous gene is enhanced by the gene recombination.
  • the recombinant cell before the introduction of the linear nucleic acid may be referred to as a host cell.
  • the host cell is not particularly limited as long as it is a cell into which a linear nucleic acid can be introduced.
  • the biological species of the recombinant cell and the host cell are not particularly limited, and examples thereof include yeast, bacteria, fungi, insect cells, animal cells and plant cells, with yeast being preferred and methanol assimilation. Sex yeast, fission yeast, and germination yeast are more preferable, and methanol-utilizing yeast is even more preferable.
  • methanol-utilizing yeast is defined as yeast that can be cultivated using methanol as the only carbon source. Originally, it was methanol-utilizing yeast, but it was assimilated into methanol by artificial modification or mutation. Yeasts that have lost their performance are also included in the methanol-utilizing yeasts of the present invention.
  • yeasts belonging to the genus Pichia, Ogataea, Komagataella, etc. yeasts of the genus Komagataella or yeasts of the genus Ogataea are preferable, and yeasts of the genus Komagataela are particularly preferable. ..
  • a strain derived from these yeast strains of the genus Komagataera can also be used, and examples thereof include Komagataera pastris GS115 strain (available from Thermo Fisher Scientific Co., Ltd.) as a histidine requirement.
  • a non-homologous recombination mechanism disrupting strain ( ⁇ ku70, ⁇ dnl4) of Komagataera fafi can also be used.
  • strains derived from these strains and the like can also be used.
  • the endogenous gene includes not only DNA but also its mRNA and cDNA among the nucleic acids possessed by the cell of the present invention, but can be typically DNA. In particular, it can be genomic DNA. Further, the endogenous gene does not ask the distinction of the functional region, and may contain, for example, only exons, or may contain exons and introns.
  • the endogenous gene is not particularly limited as long as it is a gene contained in the cell of the present invention.
  • it is composed of the following endogenous genes (1) to (32) for the purpose of enhancing the production of the target protein described later.
  • At least one endogenous gene selected from the group is exemplified.
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 47 (EF1st-3).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 49 (EF1st-5).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 50 (EF1st-6).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 51 (EF1st-7).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 52 (EF1st-8).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 53 (EF1st-9).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 54 (EF1st-10).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 55 (EF1st-11).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 56 (EF1st-12).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 57 (EF1st-13).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 58 (EF1st-14).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 59 (EF1st-15).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 60 (EF1st-16).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 61 (EF1st-17).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 63 (EF2nd-1).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 76 (EF3rd-6).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 77 (EF3rd-7).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 78 (EF3rd-8), and (32) the base represented by SEQ ID NO: 79 (EF3rd-9).
  • At least one endogenous gene selected from the group consisting of the above-mentioned endogenous genes (1) to (32) is preferably selected from the group consisting of the following (a) to (g) endogenous genes. It may be at least two endogenous genes.
  • C An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 63 (EF2nd-1).
  • the endogenous gene may further include the following endogenous genes in addition to at least one endogenous gene selected from the group consisting of the above-mentioned endogenous genes (1) to (32). (33) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 45 (EF1st-1).
  • Examples of the endogenous gene include the following combinations.
  • A An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 45 (EF1st-1).
  • B An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 45 (EF1st-1).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 66 (EF2nd-4).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 71 (EF3rd-1), and the same or substantially the same as the base sequence represented by SEQ ID NO: 75 (EF3rd-5).
  • An endogenous gene containing the same base sequence. (C) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 45 (EF1st-1).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 47 (EF1st-3).
  • D An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 66 (EF2nd-4), and the same base sequence represented by SEQ ID NO: 73 (EF3rd-3). Or an endogenous gene containing substantially the same base sequence.
  • the base sequence substantially the same as the base sequence represented by SEQ ID NOs: 45 to 71, 73, 75 to 79 is about 85% or more of the base sequence represented by SEQ ID NOs: 45 to 71, 73, 75 to 79. , Preferably about 90% or more, most preferably about 95% or more base sequences and the like.
  • identity means the optimum alignment when two base sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm is used for the optimum alignment of sequences. It means the ratio (%) of the same base sequence to the total base sequence that overlaps in (which can consider the introduction of a gap in one or both).
  • NCBI BLAST National Center for Biotechnology Information Basic Local Alignment Search Tool
  • enhanced expression of an endogenous gene means that the expression level of mRNA, which is a transcript of the endogenous gene, or polypeptide, which is a translation product, is enhanced.
  • the expression level of mRNA can be quantified by using a real-time PCR method, RNA-Seq method, Northern hybridization, hybridization method using a DNA array, or the like, and the expression level of a polypeptide recognizes a polypeptide. It can be quantified using a staining compound or the like having binding property to an antibody or a polypeptide. Further, in addition to the quantification method described above, a conventional method used by those skilled in the art may be used.
  • the degree of enhancement of the expression of the endogenous gene is not particularly limited as long as the production amount of the target protein described later is enhanced, but the transcript or translation product of the endogenous gene is not particularly limited.
  • the expression level is enhanced by 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more or 95% or more. Is preferable.
  • the method for producing a recombinant cell of the present invention includes the following steps. (1) A step of preparing a linear nucleic acid by cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme, wherein the nucleic acid fragment is a highly expressive promoter and a partial sequence starting from the stop codon of the endogenous gene. A step comprising a partial sequence into which the restriction enzyme recognition site has been inserted and a base sequence in which stop codons are sequentially linked. (2) The step of introducing the linear nucleic acid into a host cell, and (3) the endogenous gene is homologously recombined by the linear nucleic acid, and a highly expressive promoter is operably linked. A step of selecting transgenic cells containing a sex gene.
  • the method for producing a transgenic cell of the present invention includes a step (step (1)) of preparing a linear nucleic acid by cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme.
  • the nucleic acid fragment contains a highly expressive promoter, a partial sequence starting from the start codon of the endogenous gene, a partial sequence into which the restriction enzyme recognition site is inserted, and a base sequence in which stop codons are sequentially linked. It is a nucleic acid fragment.
  • the highly expressive promoter (hereinafter, the highly expressive promoter of the present invention) is not particularly limited as long as it is a promoter that enhances the expression of the endogenous gene of the cell of the present invention.
  • the type of the highly expressive promoter of the present invention may be any promoter suitable for the cells of the present invention.
  • the highly expressive promoter of the present invention is preferably the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like.
  • the cell of the present invention is a bacterium of the genus Escherichia
  • high expression promoter of the present invention trp promoter, lac promoter, recA promoter, .lambda.P L promoter, lpp promoter, T7 promoter and the like are preferable.
  • the highly expressive promoter of the present invention is preferably the SPO1 promoter, SPO2 promoter, penP promoter or the like.
  • the highly expressive promoter of the present invention is preferably an ADH promoter, a CMV (cytomegalovirus) promoter or the like.
  • the highly expressive promoter of the present invention is preferably a polyhedrin promoter, a P10 promoter or the like.
  • the highly expressive promoters of the present invention are SR ⁇ promoter, SV40 promoter, LTR promoter, CMV promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Molony mouse leukemia virus) LTR, HSV. -TK (herpes simplex virus thymidine kinase) promoter and the like are preferred.
  • the highly expressive promoter of the present invention is preferably the CaMV (cauliflower mosaic virus) 35S promoter or the like.
  • the partial sequence (hereinafter referred to as the partial sequence of the present invention) is a partial sequence starting from the start codon of the endogenous gene of the cell of the present invention and into which a restriction enzyme recognition site is inserted.
  • the nucleotide sequence information of the partial sequence starting from the start codon of the endogenous gene of the cell of the present invention can be obtained from the nucleotide sequence information described in a known database.
  • the nucleotide sequence information starting from the start codon of all endogenous genes of Komagataera fafi is the nucleotide sequence information (ACCESSION No.) of the four chromosomal DNAs of the Komagataera fafi CBS7435 strain. It can be obtained from FR839628 to FR839631 (J. Biotechnol.154 (4), 312-320 (2011)).
  • the nucleotide sequence information starting from the starting codon of the entire endogenous gene of Komagataera pastris is the nucleotide sequence information of the four chromosomal DNAs of the Komagataera pastris NBRC 0948 strain (Mattanovich). Et al., Microbial Cell Factories 8, 29 (2009)), and the nucleotide sequence information of the four chromosomal DNAs of the Komagataera Pastris GS115 strain (ACCESSION No. FN392319 to FN392322 (Nat. Biotechnol. 27 (6), 561- It can be obtained from 566 (2009))). Based on the base sequence information obtained in this way, a partial sequence starting from the start codon of the endogenous gene can be designed.
  • the length of the partial sequence of the present invention is not particularly limited as long as it is the length at which homologous recombination occurs between the endogenous gene and the linear nucleic acid, and is, for example, 20 bases or more, 50 bases or more, and 100 bases. Long or longer, 150 bases or longer.
  • the length of the partial sequence of the present invention is, for example, 1000 bases or less, 750 bases or less, 500 bases or less, or 250 bases or less.
  • the partial sequence of the present invention is a partial sequence in which a restriction enzyme recognition site is inserted.
  • the restriction enzyme recognition site is not particularly limited as long as it is a restriction enzyme recognition site present only in the partial sequence in the entire base sequence of the plasmid containing the above nucleic acid fragment.
  • Examples of restriction enzyme recognition sites inserted into the partial sequences of the present invention include sites recognized by type IIS type restriction enzymes, such as BspQI, BbsI, BsaI, and BsmBI, whose recognition sites and cleavage sites are different.
  • the above restriction enzyme recognition site may be inserted at any site within the partial sequence of the present invention, but the partial sequence of the present invention is divided to such an extent that recombination occurs homologously with the endogenous gene. It is preferably inserted in position.
  • the insertion position of the restriction enzyme recognition site in such a partial sequence is usually inserted in the middle of the partial sequence of the present invention.
  • the restriction enzyme recognition site is inserted by substituting the 92nd base.
  • the number of the restriction enzyme recognition sites inserted in the partial sequence of the present invention is not particularly limited as long as it is present only in the partial sequence in the entire base sequence of the plasmid containing the nucleic acid fragment, but is usually limited.
  • the number is one to several, preferably one or two.
  • the stop codon (hereinafter referred to as the stop codon of the present invention) is either TAA, TAG, or TGA.
  • the nucleic acid fragment (hereinafter referred to as the nucleic acid fragment of the present invention) includes a highly expressive promoter of the present invention, a partial sequence of the present invention, and a base sequence in which the stop codon of the present invention is linked in this order. That is, in the nucleic acid fragment of the present invention, the carbon at the 3'end of the highly expressive promoter of the present invention and the carbon at the 5'end of the partial sequence of the present invention form a phosphodiester bond, and the 3'of the partial sequence of the present invention. The terminal carbon and the 5'terminal carbon of the termination codon of the present invention form a phosphodiester bond.
  • a spacer sequence may be inserted between the highly expressive promoter of the present invention and the partial sequence of the present invention, and between the partial sequence of the present invention and the stop codon of the present invention.
  • the length of the spacer sequence may be appropriately determined by those skilled in the art, and may be, for example, 15 to 25 bases in length.
  • the nucleic acid fragment of the present invention uses the genomic DNA fraction prepared from the cells of the present invention as a template, and a primer is used from the base sequence information of the highly expressive promoter and the endogenous gene described in a known database. It can be designed and directly amplified by Polymerase Chain Reaction (hereinafter abbreviated as "PCR method").
  • PCR method Polymerase Chain Reaction
  • the restriction enzyme recognition site into which the partial sequence of the present invention is inserted can be introduced into the partial sequence by a known site-specific mutagenesis method.
  • the nucleic acid fragment of the present invention can be obtained by outsourcing the production to Agilent Technologies.
  • the length of the nucleic acid fragment of the present invention is not particularly limited, but is, for example, 20 bases or more, 50 bases or more, 100 bases or more, 150 bases or more, or 200 bases or more.
  • the length of the partial sequence of the present invention is, for example, 1000 bases or less, 500 bases or less, or 400 bases or less.
  • the nucleic acid fragment of the present invention is the following (i) to (xxxii). ) Is at least one nucleic acid fragment selected from the group consisting of nucleic acid fragments.
  • nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 238 (EF1st-2 OLS), (Ii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 239 (EF1st-3 OLS), (Iii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 240 (EF1st-4 OLS), (Iv) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 241 (EF1st-5 OLS), (V) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 242 (EF1st-6 OLS), (Vi) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 243 (EF1st-7 OLS), (Vii) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 244 (EF1st-8 OLS), (
  • At least one nucleic acid fragment selected from the group consisting of the above nucleic acid fragments (i) to (xxxii) is preferably selected from the group consisting of the following nucleic acid fragments (a') to (g'). It may be at least two nucleic acid fragments.
  • A' Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 239 (EF1st-3 OLS)
  • B' Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 240 (EF1st-4 OLS)
  • C' Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 255 (EF2nd-1 OLS)
  • D' Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 258 (EF2nd-4 OLS)
  • E' Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 263 (EF3rd-1 OLS)
  • nucleic acid fragment may further contain the following nucleic acid fragments in addition to at least one nucleic acid fragment selected from the group consisting of the above-mentioned nucleic acid fragments (i) to (xxxii).
  • Xxxiii A nucleic acid fragment containing the base sequence represented by SEQ ID NO: 237 (EF1st-1 OLS).
  • nucleic acid fragment examples include the following combinations.
  • A Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 237 (EF1st-1 OLS), Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 240 (EF1st-4 OLS), A nucleic acid fragment containing the base sequence represented by SEQ ID NO: 255 (EF2nd-1 OLS) and a nucleic acid fragment containing the base sequence represented by SEQ ID NO: 258 (EF2nd-4 OLS).
  • D A nucleic acid fragment containing the base sequence represented by SEQ ID NO: 258 (EF2nd-4 OLS) and a nucleic acid fragment containing the base sequence represented by SEQ ID NO: 265 (EF3rd-3 OLS).
  • a plasmid is an artificially constructed nucleic acid molecule.
  • the nucleic acid molecule that constitutes the plasmid is usually DNA, preferably double-stranded DNA.
  • plasmids include YEp vector, YRp vector, YCp vector, pPICHOLI, pHIP (Journal of General Microbioiogy (1992), 138, 2405-2416. Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha), pHRP (pHIP).
  • pHARS Molecular and General Genetics MGG February1986, Volume 202, Issue 2, pp 302-308, Transformation of the methylotrophic yeast Hansenula polymorpha by automatic replication and integration vector UC), derived from E. coli pBR322, pBluescript, pQE), plasmid vector derived from bacillus (pHY300PLK, pMTLBS72) and the like can be used.
  • the above plasmids further utilize cloning sites containing one or more restriction enzyme recognition sites, Clontech's In-Fusion cloning system, New England Biolabs' Gibson Assembly system, and the like.
  • auxotrophic complementary genes include URA3 gene, LEU2 gene, ADE1 gene, HIS4 gene, ARG4 gene and the like.
  • drug resistance genes include G418 resistance gene, Zeocin TM resistance gene, hyglomycin resistance gene, Clone NAT resistance gene, blastsaidin S resistance gene, noseoslisin resistance gene and the like.
  • step (1) the plasmid containing the nucleic acid fragment obtained as described above is cleaved with a restriction enzyme capable of cleaving the restriction enzyme recognition site contained in the partial sequence of the present invention to form a linear nucleic acid (hereinafter referred to as the present invention).
  • Linear nucleic acid is prepared.
  • the latter half of the partial sequence of the present invention (homologous sequence 1) linked to the stop codon is placed at the 5'end and highly expressed at the 3'end.
  • a linear nucleic acid can be prepared in which the first half (homologous sequence 2) of the partial sequence of the present invention linked to the sex promoter is arranged.
  • the method for producing a recombinant cell of the present invention includes a step (step (2)) of introducing the linear nucleic acid of the present invention into a host cell.
  • a method for introducing the linear nucleic acid of the present invention into a host cell that is, a transformation method
  • a known method can be appropriately used.
  • yeast cells are used as host cells
  • an electroporation method, a lithium acetate method, or a spheroplast method can be used. Examples thereof include the spheroplast method, but the method is not particularly limited thereto.
  • the method for producing a recombinant cell of the present invention is a gene recombination containing an endogenous gene in which an endogenous gene is homologously recombined by the linear nucleic acid of the present invention and a highly expressive promoter is operably linked.
  • the step of selecting cells (step (3)) is included.
  • the linear nucleic acid of the present invention introduced into the host cell is the latter half (homologous sequence 1) and 3'of the partial sequence of the present invention linked to the stop codon located at the 5'end.
  • Homologous recombination single crossover recombination
  • the first half homologous sequence 2 of the partial sequence of the present invention linked to the highly expressive promoter located at the end as a homologous region for the endogenous gene.
  • the linear nucleic acid of the present invention is inserted between the homologous sequence 1 and the homologous sequence 2 contained in the endogenous gene, and the endogenous gene originally provided in the host cell is the linear of the present invention.
  • step (3) when a transgenic cell containing an endogenous gene operably linked to a highly expressive promoter (hereinafter referred to as the recombinant cell of the present invention) is selected, a auxotrophic complementary gene or drug resistance is selected. It is preferable to use a selectable marker gene such as a gene.
  • the selection marker is not particularly limited, but if the host cell is yeast of the genus Komagataera, if it is an auxotrophic complementary gene such as URA3 gene, LEU2 gene, ADE1 gene, HIS4 gene, ARG4 gene, uracil, leucine, adenin, respectively.
  • the genetically modified cells of the present invention can be selected by restoring the auxotrophic strain phenotype in the auxotrophic strains of yeast and arginine.
  • it is a drug resistance gene such as G418 resistance gene, Zeocin TM resistance gene, Hyglomycin resistance gene, Clone NAT resistance gene, Blasticidin S resistance gene, G418, Zeocin TM, Hyglomycin, Clone, respectively.
  • the recombinant cells of the present invention can be selected by resistance on a medium containing NAT and Blasticidin S.
  • the auxotrophic selectable marker used when producing the recombinant yeast cannot be used if the selectable marker is not destroyed in the host yeast. In this case, the selectable marker may be destroyed in the host yeast, and a method known to those skilled in the art can be used as the method.
  • the present invention provides transgenic cells with enhanced expression of endogenous genes (hereinafter, the recombinant cells of the present invention).
  • the genetically modified cell of the present invention can be prepared by the method for producing a genetically modified cell of the present invention.
  • the recombinant cell of the present invention can function as a highly expressive promoter in which an endogenous gene is homologously recombined with a linear nucleic acid in which a plasmid containing a nucleic acid fragment is cleaved with a limiting enzyme.
  • the nucleic acid fragment is a highly expressive promoter, a partial sequence starting from the starting codon of the endogenous gene, and the restriction enzyme recognition site is inserted.
  • a recombinant cell containing a base sequence in which a partial sequence and a termination codon are sequentially linked.
  • the gene-recombinant cell, endogenous gene, nucleic acid fragment, plasmid, restriction enzyme, highly expressive promoter, partial sequence, etc. in the gene-recombinant cell of the present invention are described in the method for producing a gene-recombinant cell of the present invention. May be the same as.
  • the recombinant cell of the present invention may contain a base sequence encoding a target protein in its genome.
  • the target protein is a protein produced by a cell whose genome contains a base sequence encoding the target protein, and may be an endogenous protein of the cell or a heterologous protein.
  • the target protein include enzymes derived from microorganisms, proteins produced by animals and plants that are multicellular organisms, and the like.
  • Examples thereof include phytase, protein A, protein G, protein L, amylase, glucosidase, cellulase, lipase, protease, glutaminase, peptidase, nuclease, oxidase, lactase, xylanase, trypsin, pectinase, isomerase, fibroin, fluorescent protein and the like.
  • phytase protein A, protein G, protein L, amylase, glucosidase, cellulase, lipase, protease, glutaminase, peptidase, nuclease, oxidase, lactase, xylanase, trypsin, pectinase, isomerase, fibroin, fluorescent protein and the like.
  • human and / or animal therapeutic proteins are preferred.
  • hepatitis B virus surface antigen As proteins for human and / or animal treatment, specifically, hepatitis B virus surface antigen, hirudin, antibody, human antibody, partial antibody, human partial antibody, serum albumin, human serum albumin, epithelial growth factor, human epithelial growth.
  • Factors insulin, growth hormone, erythropoetin, interferon, blood coagulation factor VIII, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), thrombopoetin, IL-1, IL-6, Tissue plasminogen activator (TPA), urokinase, leptin, stem cell growth factor (SCF) and the like.
  • G-CSF granulocyte colony stimulating factor
  • GM-CSF granulocyte macrophage colony stimulating factor
  • TPA Tissue plasminogen activator
  • urokinase urokinase
  • the antibody refers to a heterotetramer protein composed of two polypeptide chains, an L chain and an H chain, formed by disulfide bonds, especially if it has the ability to bind to a specific antigen. Not limited.
  • the partial antibody refers to a Fab antibody, (Fab) 2 antibody, scFv antibody, diabody antibody, camel VHH antibody, derivatives thereof, etc., as long as it has the ability to bind to a specific antigen.
  • Fab antibody refers to a heteromer protein in which the L chain and Fd chain of an antibody are bound by an SS bond, or a heteromer protein in which the L chain and Fd chain of an antibody are associated without an SS bond. It is not particularly limited as long as it has the ability to combine.
  • amino acids constituting the above-mentioned target protein may be natural, non-natural, or modified.
  • amino acid sequence of the protein may be artificially modified or may be designed by de-novo.
  • the base sequence encoding the above-mentioned target protein is contained in an expression vector, and is integrated by homologous recombination into an arbitrary site in the genome of the recombinant cell of the present invention.
  • the expression vector can be produced, for example, by cutting out a DNA fragment containing a base sequence encoding the above-mentioned target protein and linking the DNA fragment downstream of a promoter in an appropriate expression vector.
  • Expression vectors include Escherichia coli-derived plasmids (eg, pBR322, pBR325, pUC12, pUC13); Bacteriophage-derived plasmids (eg, pUB110, pTP5, pC194); Yeast-derived plasmids (eg, pSH19, pSH15); Insect cell expression.
  • Escherichia coli-derived plasmids eg, pBR322, pBR325, pUC12, pUC13
  • Bacteriophage-derived plasmids eg, pUB110, pTP5, pC194
  • Yeast-derived plasmids eg, pSH19, pSH15
  • Insect cell expression include Escherichia coli-derived plasmids (eg, pBR322, pBR325, pUC12, pUC13); Bacteriophage-derived plasmids
  • Plasmid eg pFast-Bac
  • animal cell expression plasmid eg pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo
  • bacteriophage such as ⁇ phage
  • insect virus vector such as baculovirus (eg) Example: BmNPV, AcNPV)
  • Animal virus vectors such as retrovirus, vaccinia virus, and adenovirus are used.
  • the promoter may be any promoter as long as it is suitable for the host used for gene expression, and for example, the highly expressive promoter of the present invention is preferably mentioned.
  • a vector containing an enhancer, a splicing signal, a poly A addition signal, a selectable marker, an SV40 origin of replication, or the like can be used, if desired.
  • the selectable marker include a dihydrofolate reductase (dhfr) gene, an ampicillin resistance gene, a neomycin resistance gene and the like.
  • dhfr gene-deficient Chinese hamster cells when dhfr gene-deficient Chinese hamster cells are used and the dhfr gene is used as a selectable marker, the target gene can also be selected using a thymidine-free medium.
  • a base sequence (signal codon) encoding a signal sequence suitable for the host cell is added (or replaced with a native signal codon) to the 5'end side of the base sequence encoding the target protein. May be good.
  • the host cell is a bacterium of the genus Escherichia, the PhoA signal sequence, the OmpA signal sequence, etc .; if the host cell is a bacterium of the genus Bacillus, the ⁇ -amylase signal sequence, the subtilisin signal sequence, etc.; If the host cell is an animal cell, the insulin signal sequence, ⁇ -interferon signal sequence, antibody molecule signal sequence, etc. are used, respectively.
  • the expression vector is a partial sequence of the genome of the recombinant cell of the present invention, and contains a partial sequence in which a restriction enzyme recognition site is inserted.
  • the linear expression vector cleaved by the restriction enzyme was introduced into the recombinant cell of the present invention and placed at the latter half (homologous sequence 1) and 3'end of the partial sequence arranged at the 5'end.
  • Homologous recombination single crossover recombination
  • the base sequence encoding the target protein of the present invention is inserted into the genome of the recombinant cell of the present invention.
  • a method for producing a target protein (hereinafter, a method for producing a target protein of the present invention), which comprises a step of culturing a recombinant cell of the present invention containing a base sequence encoding the target protein in the genome.
  • the cell culture conditions are not particularly limited and may be appropriately selected according to the cells.
  • any medium containing a nutrient source capable of assimilating cells can be used.
  • the nutrient source include sugars such as glucose, shoe cloth and maltose, organic acids such as lactic acid, acetic acid, citric acid and propionic acid, alcohols such as methanol, ethanol and glycerol, hydrocarbons such as paraffin and soybean oil. Oils such as rapeseed oil, carbon sources such as mixtures thereof, nitrogen sources such as ammonium sulfate, ammonium phosphate, urea, yeast extract, meat extract, peptone, corn steep liquor, and other inorganic salts, vitamins, etc.
  • a normal medium in which nutrient sources are appropriately mixed can be used.
  • the culture can be either batch culture or continuous culture.
  • the carbon source may be one kind of glucose, glycerol, and methanol, or two or more kinds. Further, these carbon sources may be present from the initial stage of culturing, or may be added during culturing.
  • the target protein By culturing the recombinant cell of the present invention containing the base sequence encoding the target protein in the genome, the target protein can be accumulated and recovered in the cell or in the culture medium.
  • a known purification method can be used in an appropriate combination. For example, first, the recombinant cells of the present invention containing the nucleotide sequence encoding the target protein in the genome are cultured in an appropriate medium, and the cells are removed from the culture supernatant by centrifugation or filtration of the culture medium.
  • the obtained culture supernatant is subjected to salting (ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (protein fractionation precipitation method using acetone, ethanol, etc.), dialysis, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography.
  • the target protein is recovered from the culture supernatant by using techniques such as imaging, affinity chromatography, reverse phase chromatography, and ultrafiltration alone or in combination.
  • the cells can usually be cultured under general conditions.
  • the cells are aerobically cultured for 10 hours to 10 days in a pH range of 2.5 to 10.0 and a temperature range of 10 ° C to 48 ° C. It can be done by.
  • the recovered target protein can be used as it is, but it can also be used afterwards with modifications that bring about pharmacological changes such as PEGylation and modifications that add functions such as enzymes and isotopes. Moreover, various formulation treatments may be used.
  • the endogenous gene originally provided in the host cell loses its function due to the stop codon contained in the linear nucleic acid of the present invention. Instead, downstream of the dysfunctional gene results in a new gene (the same gene as the endogenous gene) operably linked to the highly expressive promoter contained in the linear nucleic acid of the invention.
  • the expression of the endogenous gene is enhanced by the highly expressive promoter. Therefore, the recombinant cell population of the present invention in which the expression of each gene of all endogenous genes of the host cell is enhanced can be used as a cell library in which the expression of each endogenous gene is enhanced.
  • the screening method 1 of the present invention includes the following steps. (1) A step of preparing a linear nucleic acid by cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme, wherein the nucleic acid fragment is a highly expressive promoter and a partial sequence starting from the stop codon of the endogenous gene. A step comprising a partial sequence into which the restriction enzyme recognition site has been inserted and a base sequence in which stop codons are sequentially linked. (2) A step of introducing the linear nucleic acid into a host cell containing a base sequence encoding a target protein in the genome.
  • the screening method 1 of the present invention is a step of preparing a linear nucleic acid by cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme, wherein the nucleic acid fragment is a highly expressive promoter and a stop codon of the endogenous gene.
  • the step (step (1)) is included, which comprises a partial sequence starting from, a partial sequence into which the restriction enzyme recognition site is inserted, and a base sequence in which stop codons are sequentially linked.
  • the step (1) in the screening method 1 of the present invention may be the same as the step (1) in the method for producing a recombinant cell of the present invention.
  • the screening method 1 of the present invention is a step of introducing the linear nucleic acid of the present invention into a host cell (hereinafter, a cell expressing the target protein of the present invention) containing a base sequence encoding the target protein in the genome (step (2)). )including.
  • the method for introducing the linear nucleic acid of the present invention into the target protein-expressing cell of the present invention may be the same as that described in step (2) in the method for producing a recombinant cell of the present invention.
  • the target protein may be the same as the target protein contained in the genome of the recombinant cell of the present invention.
  • the host cell may be the same as the host cell used in the method for producing a recombinant cell of the present invention.
  • the screening method 1 of the present invention selects transgenic cells containing an endogenous gene operably linked with a highly expressive promoter, in which the endogenous gene is homologously recombined by the linear nucleic acid of the present invention. Includes step (step (3)).
  • the step (3) in the screening method 1 of the present invention may be the same as the step (3) in the method for producing a recombinant cell of the present invention.
  • the screening method 1 of the present invention includes a step (step (4)) of culturing the cells obtained in the step (3) and the target protein-expressing cells of the present invention.
  • the method for culturing the cells obtained in step (3) and the target protein-expressing cells of the present invention may be the same as the culturing method described in the method for culturing the recombinant cells of the present invention.
  • the screening method 1 of the present invention includes a step (step (5)) of measuring the production amount of the target protein by the cells obtained in the step (3) and the target protein-expressing cells, respectively.
  • the amount of target protein produced by the cells obtained in step (3) and the target protein-expressing cells of the present invention is measured by measuring the expression level of mRNA, which is a transcript of the target protein, or polypeptide, which is a translation product. It can be carried out.
  • the expression level of mRNA can be quantified by using real-time PCR method, RNA-Seq method, Northern hybridization, hybridization method using DNA array, etc., and the expression level of polypeptide can be determined by an antibody that recognizes a polypeptide or It can be quantified using a staining compound or the like having binding property to the polypeptide. Further, in addition to the quantification method described above, a conventional method used by those skilled in the art may be used.
  • the screening method 1 of the present invention includes a step (step (6)) of identifying an endogenous gene that increases the production amount of the target protein.
  • step (6) if the production amount of the target protein in the cells obtained in step (3) is higher than the production amount of the target protein in the target protein-expressing cells of the present invention, the cells increase the production amount of the target protein. It can be determined that the cell contains an endogenous gene.
  • the increase in the production amount of the target protein in the cells obtained in step (3) is, for example, 1.01 times, 1.02 times, 1.03 times, 1.04 times, 1.05 times the production amount of the target protein in the target protein-expressing cells of the present invention.
  • the amount of total protein secreted and produced from the cells can be easily determined by a method known to those skilled in the art, such as the Bladeford method, the Lowry method, the BCA method, etc., using the cell culture supernatant or the like. Can be decided.
  • the amount of secreted production of a specific target protein from cells can be easily determined by an ELISA method or the like using a cell culture supernatant or the like.
  • the endogenous gene whose expression is enhanced by a known means.
  • the genome is extracted from cells, fragmented with a restriction enzyme, and then the fragmented genome is self-ligated. Genome fragments containing the endogenous gene of interest cyclized by self-ligation can be screened by drug resistance genes.
  • the endogenous gene can be specifically identified by specifying the nucleotide sequence of the screened genomic fragment containing the target endogenous gene by the Sanger method.
  • the screening method 2 of the present invention includes the following steps. (1) A step of introducing an expression vector containing a base sequence encoding a target protein into a cell library overexpressing an endogenous gene of the present invention and a host cell and culturing the cells. (2) A step of measuring the production amount of the target protein by the endogenous gene overexpressing cell library and the host cell, and (3) a step of identifying the endogenous gene that increases the production amount of the target protein.
  • the screening method 2 of the present invention includes a step (step (1)) of introducing an expression vector containing a base sequence encoding a target protein into a host cell and an endogenous gene overexpressing cell library of the present invention and culturing the cells.
  • the method of introducing the endogenous gene overexpressing cell library of the present invention and the expression vector containing the base sequence encoding the target protein into the host cell and culturing the method is the method of introducing the linear nucleic acid of the present invention into the host cell and the present invention. It may be the same as that described in the method for culturing transgenic cells of the present invention.
  • the screening method 2 of the present invention includes a step (step (2)) of measuring the production amount of the target protein by the endogenous gene overexpressing cell library and the host cell.
  • the method for measuring the production amount of the target protein by the endogenous gene overexpressing cell library and the host cell may be the same as the method for measuring the production amount of the target protein in the screening method 1 of the present invention.
  • the screening method 2 of the present invention includes a step (step (3)) of identifying an endogenous gene that increases the production of the target protein.
  • the method for identifying the endogenous gene that increases the production of the target protein may be the same as the method for identifying the endogenous gene in the screening method 1 of the present invention.
  • GAP promoter SEQ ID NO: 1
  • AOX1 promoter SEQ ID NO: 2
  • AOX1 terminator SEQ ID NO: 3
  • CCA38473 terminator SEQ ID NO: 4
  • ARG4 gene SEQ ID NO: 5
  • the downstream sequence (SEQ ID NO: 6), GUT1 gene (SEQ ID NO: 7), the gene encoding KAR2 (SEQ ID NO: 8) derived from Komagataera fafi to which the terminator is linked, and the gene encoding PDI1 (SEQ ID NO: 9) are Komagataera.
  • the chromosomal DNA of the Faffy CBS7435 strain (the base sequence is described in EMBL (The European Molecular Biology Laboratory) ACCESSION No. FR839628 to FR839631) was prepared by PCR using a mixture as a template.
  • the gene (SEQ ID NO: 45-79) encoding the antibody expression-promoting protein group (amino acid sequence shown by SEQ ID NOs: 10 to 44) derived from Komagataera fafi to which the terminator is linked is the genome of Komagataera fafi obtained by screening. Prepared by PCR as a template.
  • the GAP promoter is Primer 1 (SEQ ID NO: 80) and Primer 2 (SEQ ID NO: 81)
  • the AOX1 promoter is Primer 3 (SEQ ID NO: 82) and Primer 4 (SEQ ID NO: 83)
  • the AOX1 terminator is Primer 5 (SEQ ID NO: 84) and Primer.
  • CCA38473 terminator is primer 7 (SEQ ID NO: 86) and primer 8 (SEQ ID NO: 87), promoter-regulated ARG4 gene is primer 9 (SEQ ID NO: 88) and primer 10 (SEQ ID NO: 89),
  • the URA3 gene lacking the starting codon and having the AscI-PmeI recognition site added inside is Primer 11 (SEQ ID NO: 90), Primer 12 for mutagenesis (SEQ ID NO: 91), and Primer 13 for mutagenesis (SEQ ID NO: 92).
  • primer 14 (SEQ ID NO: 93), the GUT1 gene lacking the starting codon and having the AscI-PmeI recognition site added internally is primer 15 (SEQ ID NO: 94) and primer 16 for mutagenesis (SEQ ID NO: 95),
  • the gene encoding the mutation-introducing primer 17 (SEQ ID NO: 96) and primer 18 (SEQ ID NO: 97), and the antibody expression-promoting protein (amino acid sequence shown by SEQ ID NOs: 10 to 44) to which the terminator is linked is a primer for forward. It was prepared by PCR using 19 (SEQ ID NO: 98) and primers 20 to 55 (SEQ ID NO: 99 to 134) for each reverse.
  • the secretory signal MF ⁇ gene (SEQ ID NO: 135) used in the construction of the vector is the chromosomal DNA of the Saccharomyces cerevisiae BY4741 strain (the base sequence is described in ACCESSION No. BK006934 to BK006949). It was prepared by PCR using Primer 57 (SEQ ID NO: 137).
  • the diamino acid substitution (L42S / V50A) (SEQ ID NO: 138) of the secretory signal MF ⁇ gene was prepared by PCR using synthetic DNA as a template and primers 56 (SEQ ID NO: 136) and 57 (SEQ ID NO: 137).
  • the promoter-controlled Zeocin TM resistance gene (SEQ ID NO: 139) used in the construction of the vector was prepared by PCR using synthetic DNA as a template.
  • the promoter-regulated G418 resistance gene (SEQ ID NO: 140) used in the construction of the vector was prepared by PCR using synthetic DNA as a template.
  • the promoter-controlled hygromycin resistance gene (SEQ ID NO: 141) used in the construction of the vector was prepared by PCR using synthetic DNA as a template.
  • the promoter-regulated noseoslisin resistance gene (SEQ ID NO: 142) used in the construction of the vector was prepared by PCR using synthetic DNA as a template.
  • the promoter-controlled blastidin resistance gene (SEQ ID NO: 143) used in the construction of the vector was prepared by PCR using synthetic DNA as a template.
  • the anti-lysothium single-chain antibody gene (SEQ ID NO: 144), tandem scFv226 antibody gene (SEQ ID NO: 145), blinatumomab antibody gene (SEQ ID NO: 146) and minibody antibody (SEQ ID NO: 147) used in the construction of the vector are synthetic DNAs. Was used as a template and prepared by PCR.
  • the KAR2 gene (SEQ ID NO: 8) and PDI1 gene (SEQ ID NO: 9) used in the construction of the vector were prepared by PCR using the genome of the Komagataera fafi CBS7435 strain as a template.
  • Prime STAR HS DNA Polymerase (manufactured by Takara Bio Inc.) was used for PCR, and the reaction conditions were as described in the attached manual.
  • the chromosomal DNA was prepared from the Komagataera pastris ATCC76273 strain or the Saccharomyces cerevisiae BY4741 strain using Kaneka Simple DNA Extraction Kit version 2 (manufactured by Kaneka Corporation) under the conditions described therein.
  • pUC_G418 a nucleic acid fragment of CCA38473 terminator (SEQ ID NO: 4) was prepared by PCR using Primer 7 (SEQ ID NO: 86) and Primer 8 (SEQ ID NO: 87), and the above pUC_G418 was mixed with the XbaI-treated nucleic acid fragment and In. -pUC_T38473_G418 was constructed by connecting using the fusion HD Cloning Kit (manufactured by Clontech).
  • nucleic acid fragment having a BamHI recognition sequence and a SpeI recognition sequence added to the end of the AOX1 promoter (SEQ ID NO: 2) was prepared by PCR using Primer 3 (SEQ ID NO: 82) and Primer 4 (SEQ ID NO: 83). BamHI and SpeI treatment, pUC_T38473_G418 was inserted between the BamHI-BglII sites to construct pUC_Paox1_T38473_G418.
  • nucleic acid fragment having the MluI recognition sequence and the BglII recognition sequence added to the end of the AOX1 terminator (SEQ ID NO: 3) was prepared by PCR using Primer 5 (SEQ ID NO: 84) and Primer 6 (SEQ ID NO: 85).
  • pUC_Paox1_T38473_G418 was inserted between the MluI-BglII sites to construct pUC_Paox1_Taox1_T38473_G418.
  • nucleic acid fragment having a SpeI recognition sequence and a BglII recognition sequence added to the ends of the secretory signal MF ⁇ gene (SEQ ID NO: 135) and its two amino acid substitutions (L42S / V50A) (SEQ ID NO: 138) was added to Primer 56 (SEQ ID NO: 136).
  • primer 57 SEQ ID NO: 137, respectively, after treatment with SpeI and BglII, each was inserted between the MluI-BglII sites of pUC_Paox1_Taox1_T38473_G418 to construct pUC_Paox1_MF ⁇ _Taox1_T38473_G418 and pUC_Paox1_MF ⁇ (mut) _43.
  • nucleotide sequence encoding a His tag (SEQ ID NO: 153) is added between the nucleotide sequence encoding the anti-lysozyme antibody and the upstream terminal region of the AOX1 terminator sequence.
  • the pUC_Pgap_MF ⁇ _Taox1_T38473_G418 constructed in (2) above was treated with XhoI and MluI to prepare a nucleic acid fragment, which was mixed with the nucleic acid fragment having the nucleotide sequence encoding the anti-lysodium single-chain antibody prepared by the above PCR, and the In-fusion HD Cloning Kit was used.
  • Bispecific antibody tandem sc Fv226 expression vector Bispecific antibody (tandem scFv226; taFv226) gene (SEQ ID NO:) in which an anti-CD3 single-chain antibody and an anti-EGFR single-chain antibody are fused. 145) was prepared by PCR using synthetic DNA as a template and Primer 62 (SEQ ID NO: 154) and Primer 63 (SEQ ID NO: 155).
  • This nucleic acid fragment is the downstream terminal region of the secretory signal MF ⁇ gene sequence (2-amino acid substitution (L42S / V50A)) as an overlapping region upstream of the base sequence encoding the taFv226 antibody, and upstream of the AOX1 terminator sequence as an overlapping region downstream.
  • the terminal region is added.
  • a nucleotide sequence encoding a c-Myc tag (SEQ ID NO: 156) and a His tag (SEQ ID NO: 153) are added between the nucleotide sequence encoding the taFv226 antibody and the upstream terminal region of the AOX1 terminator sequence.
  • the pUC_Paox1_MF ⁇ (mut) _Taox1_T38473_G418 constructed in (2) above was treated with XhoI and MluI to prepare a nucleic acid fragment, which was mixed with the nucleic acid fragment having the nucleotide sequence encoding the bispecific antibody (taFv226) prepared by PCR above.
  • a nucleic acid fragment of CCA38473 terminator (SEQ ID NO: 4) was prepared by PCR using primer 64 (SEQ ID NO: 157) and primer 65 (SEQ ID NO: 158), and the above pUC_Paox1_MF ⁇ (mut) _taFv226_Taox1_T38473_G418 was treated with the nucleic acid fragment treated with XbaI.
  • the mixture was mixed and spliced using an In-fusion HD Cloning Kit (manufactured by Clontech) to construct pUC_Paox1_MF ⁇ (mut) _taFv226_Taox1_loxP_T38473_loxP_G418.
  • This vector is designed for bispecific antibody (taFv226) to be expressed under AOX1 promoter control.
  • the blinatumomab gene (SEQ ID NO: 146), which is the only bispecific low molecular weight antibody currently on the market, is used as a template for primer 66 (SEQ ID NO: 159) and primer. Prepared by PCR using 67 (SEQ ID NO: 160).
  • This nucleic acid fragment is the downstream terminal region of the secretory signal MF ⁇ gene sequence (2-amino acid substituent (L42S / V50A)) as an overlapping region upstream of the nucleotide sequence encoding the brinatsumomab antibody, and upstream of the AOX1 terminator sequence as an overlapping region downstream. The terminal region is added.
  • nucleotide sequence encoding a His tag (SEQ ID NO: 153) is added between the nucleotide sequence encoding the blinatumomab antibody and the upstream terminal region of the AOX1 terminator sequence.
  • the pUC_Paox1_MF ⁇ (mut) _taFv226_Taox1_loxP_T38473_loxP_G418 constructed in (4) above was treated with XhoI and MluI to prepare a nucleic acid fragment, mixed with the nucleic acid fragment having the nucleotide sequence encoding the blinatumomab antibody prepared by the above PCR, and the In-fusion HD Cloning Kit.
  • minibody antibody expression vector Primer 68 of the minibody gene (SEQ ID NO: 147), which is a small antibody fused with an anti-EGFR single-chain antibody gene and a part of the Fc region, using synthetic DNA as a template. It was prepared by PCR using (SEQ ID NO: 161) and primer 69 (SEQ ID NO: 162). This nucleic acid fragment is the downstream terminal region of the secretory signal MF ⁇ gene sequence (2-amino acid substituent (L42S / V50A)) as an overlapping region upstream of the nucleotide sequence encoding the minibody antibody, and the AOX1 terminator sequence downstream as an overlapping region. The upstream end region is added.
  • a nucleotide sequence encoding a His tag (SEQ ID NO: 153) is added between the nucleotide sequence encoding the minibody antibody and the upstream terminal region of the AOX1 terminator sequence.
  • Nucleic acid fragments were prepared by treating pPGP_EGFP with BamHI and SpeI, mixed with the two nucleic acid fragments prepared by the above PCR, and spliced using the In-fusion HD Cloning Kit to prepare pPGPdel_EGFP.
  • a nucleic acid fragment was prepared by PCR using primer 76 (SEQ ID NO: 169) and primer 77 (SEQ ID NO: 170) using pPGPdel_EGFP as a template. ..
  • nucleic acid fragment of CYC1 terminator (SEQ ID NO: 171) derived from Saccharomyces cerevisiae
  • PCR using Saccharomyces cerevisiae BY4741 strain as a template and Primer 78 (SEQ ID NO: 172) and Primer 79 (SEQ ID NO: 173) was performed.
  • Nucleic acid fragments were prepared after treating pUC_del_Zeo with SacI and BamHI, mixed with the two nucleic acid fragments prepared by the above PCR, and spliced using the In-fusion HD Cloning Kit to prepare pUC_del_Zeo_Pgap-EGFP-CYC1t.
  • this vector can be used as a vector for producing a gene overexpressing cell library using Komagataera fafi. Is designed for.
  • KAR2 and PDI1 gene overexpression vector Primer 70 (SEQ ID NO: 163) and primer were added to the nucleic acid fragment in which the EcoRI recognition sequence was added to both ends of the Zeocin TM resistance gene (SEQ ID NO: 139) controlled by the promoter. It was prepared by PCR using 71 (SEQ ID NO: 164) and inserted between the EcoRI sites of pUC19 to construct pUC_Zeo.
  • nucleic acid fragments of the first half and the second half of the ARG4 gene (SEQ ID NO: 5) regulated by the promoter are subjected to Primer 9 (SEQ ID NO: 88), Primer 80 for mutation insertion (SEQ ID NO: 174), and Primer 81 (SEQ ID NO: 81).
  • Primer 9 SEQ ID NO: 88
  • Primer 80 for mutation insertion SEQ ID NO: 174
  • Primer 81 SEQ ID NO: 81
  • a pUC_Arg4_Zeo having a sequence into which a PmeI restriction enzyme site (SEQ ID NO: 176) was introduced was constructed.
  • a nucleic acid fragment having a HindIII recognition sequence added to one end of the EGFP expression cassette (GAPDH promoter-EGFP gene-AOX1t) was added to pPGP_EGFP (Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018).
  • nucleic acid fragment in which the SpeI recognition sequence and the HindIII recognition sequence were added to both ends of the gene encoding the KAR2 and PDI1 proteins to which the terminator was ligated was added to Primer 84 (SEQ ID NO: 181) and Prepared by PCR using Primer 85 (SEQ ID NO: 182), Primer 86 (SEQ ID NO: 183) and Primer 87 (SEQ ID NO: 184), mixed with SpeI and HindIII treated pUC_Arg4_Pgap_EGFP_Taox1_Zeo, and used with In-fusion HD Cloning Kit.
  • This vector is designed to overexpress KAR2 or PDI1 as a model protein under the control of the GAPDH promoter.
  • pUC_Arg4_Pgap_EF3rd-9_T37552_Zeo 36 types of pUC_Arg4_Pgap_EF3rd-9_T37552_Zeo by connecting them using the In-fusion HD Cloning Kit.
  • This vector is designed so that antibody production promoting proteins (amino acid sequences shown in SEQ ID NOs: 10 to 44) are each expressed under the control of the GAPDH promoter.
  • nucleic acid fragments of the first half and the second half of the URA3 gene (SEQ ID NO: 6) from which the starting codon was deleted were subjected to primer 90 (SEQ ID NO: 187), primer 91 for mutagenesis (SEQ ID NO: 188), and mutagenesis, respectively.
  • primer 90 SEQ ID NO: 187
  • primer 91 for mutagenesis SEQ ID NO: 188
  • mutagenesis respectively.
  • Primer 92 SEQ ID NO: 189
  • Primer 93 SEQ ID NO: 190
  • the SpeI recognition sequence and the HindIII recognition sequence were added to both ends of the gene (SEQ ID NO: 45 to 52) encoding the antibody production promoting protein (8 kinds of amino acid sequences shown in SEQ ID NOs: 10 to 17) to which the terminator was linked.
  • Nucleic acid fragments were prepared by PCR using forward primer 19 (SEQ ID NO: 98) and their respective reverse primers 20-27 (SEQ ID NO: 99-106), mixed with SpeI and HindIII treated pUC_URA3_Pgap_EGFP_Taox1_Hyg and mixed.
  • pUC_URA3_Pgap_EF2nd-4_Hyg Eight types of pUC_URA3_Pgap_EF2nd-4_Hyg were constructed from pUC_URA3_Pgap_EF1st-1_Hyg by connecting them using the In-fusion HD Cloning Kit.
  • This vector is designed so that antibody production promoting proteins (8 kinds of amino acid sequences shown in SEQ ID NOs: 10 to 17) are expressed under the control of the GAPDH promoter.
  • nucleic acid fragments of the first half and the second half of the GUT1 gene (SEQ ID NO: 7) from which the starting codon was deleted were used as primer 95 (SEQ ID NO: 193), primer 96 for mutagenesis (SEQ ID NO: 194), and mutagenesis, respectively.
  • primer 95 SEQ ID NO: 193
  • primer 96 for mutagenesis SEQ ID NO: 194
  • mutagenesis respectively.
  • pUC_Gut1_Pgap_EGFP_Taox1_NAT having a sequence in which the AscI-PmeI restriction enzyme site (SEQ ID NO: 191) was introduced in the center of the downstream sequence (0.7Kb).
  • the SpeI recognition sequence and the HindIII recognition sequence were added to both ends of the gene (SEQ ID NO: 45 to 52) encoding the antibody production promoting protein (8 kinds of amino acid sequences shown in SEQ ID NOs: 10 to 17) to which the terminator was linked.
  • Nucleic acid fragments were prepared by PCR using forward primer 19 (SEQ ID NO: 98) and their respective reverse primers 20-27 (SEQ ID NO: 99-106), mixed with SpeI and HindIII treated pUC_Gut1_Pgap_EGFP_Taox1_NAT and mixed.
  • pUC_GUT1_Pgap_EF2nd-4_NAT Eight types of pUC_GUT1_Pgap_EF2nd-4_NAT were constructed from pUC_GUT1_Pgap_EF1st-1_NAT by connecting them using the In-fusion HD Cloning Kit.
  • This vector is designed so that antibody production promoting proteins (8 kinds of amino acid sequences shown in SEQ ID NOs: 10 to 17) are expressed under the control of the GAPDH promoter.
  • nucleic acid fragment of the AOX1 promoter (SEQ ID NO: 2) was prepared by PCR using Primer 100 (SEQ ID NO: 198) and Primer 101 (SEQ ID NO: 199), mixed with NheI and PstI-treated pUC_Arg4_Pgap_EGFP_Taox1_bsd, and In- By connecting using the fusion HD Cloning Kit, pUC_Paox1_Pgap_EGFP_Taox1_bsd having the AOX1 promoter sequence as the genome transfer site was constructed.
  • the SpeI recognition sequence and the HindIII recognition sequence were added to both ends of the gene (SEQ ID NO: 45 to 52) encoding the antibody production promoting protein (8 kinds of amino acid sequences shown in SEQ ID NOs: 10 to 17) to which the terminator was linked.
  • Nucleic acid fragments were prepared by PCR using forward primers 19 (SEQ ID NO: 98) and their respective reverse primers 20-27 (SEQ ID NOs: 99-106) and mixed with SpeI and HindIII treated pUC_Paox1_Pgap_EGFP_Taox1_bsd.
  • pUC_Paox1_Pgap_EF2nd-4_bsd Eight types were constructed from pUC_Paox1_Pgap_EF1st-1_bsd by connecting them using the In-fusion HD Cloning Kit.
  • This vector is designed so that antibody production promoting proteins (8 kinds of amino acid sequences shown in SEQ ID NOs: 10 to 17) are expressed under the control of the GAPDH promoter.
  • Blinatumomab antibody expression vector pUC_Paox1_MF ⁇ (mut) _Blinatumomab_Taox1_loxP_T38473_loxP_G418 and the minibody antibody vector pUC_Paox1_MF ⁇ (mut) _Minibody_Taox1_loxP_T38473_loxP_G418 constructed in (6) above.
  • Komagataera fafi Dnl4 deficient / histidine auxotrophic strain (Ito et al., FEMS Yeast Research, Vol. 18, No.
  • YPD medium 1% dried yeast extract (manufactured by Nacalai Tesque), 2% Bacto Pepton (Manufactured by Becton Dickinson), 2% glucose) 2 ml, shake-cultured at 30 ° C. for 16 hours, subcultured in fresh YPD medium at 10-fold dilution, and further shake-cultured at 30 ° C. for 4 hours. After culturing yeast cells are collected by centrifugation, the yeast cells are washed (suspended by adding 6 ml of sterilized water, and the yeast cells are collected by centrifugation), and then the yeast cells are re-suspended with the sterilized water remaining on the test tube wall.
  • YPD medium 1% dried yeast extract (manufactured by Nacalai Tesque), 2% Bacto Pepton (Manufactured by Becton Dickinson), 2% glucose) 2 ml
  • yeast cells are collected, suspended in 500 ⁇ l of YPD medium (1% dried yeast extract (manufactured by Nacalai Tesque), 2% Bacto Pepton (manufactured by Becton Dickinson), 2% glucose), and then 30 The mixture was allowed to stand at ° C for 2 hours. After standing for 2 hours, yeast cells were subjected to YPDG418 selection agar plate (1% dried yeast extract (manufactured by Nakaraitesk), 2% Bacto Pepton (manufactured by Becton Dickinson), 2% glucose, 2% agarose, 0.05% G418 disulfate.
  • YPD medium 1% dried yeast extract (manufactured by Nacalai Tesque), 2% Bacto Pepton (manufactured by Becton Dickinson), 2% glucose, 2% agarose, 0.05% G418 disulfate.
  • a strain that is applied to salt (manufactured by Nakaraitesk Co., Ltd.) and grows in a static culture at 30 ° C. for 3 days is selected, and anti-lysodium single-stranded antibody-expressing yeast, tandem scFv226 antibody-expressing yeast, brinatsumomab antibody-expressing yeast, and Minibody antibody-expressing yeast was obtained.
  • Gene activation was confirmed by PCR using the chromosomal DNA of transformed yeast as a template for fragment length and internal sequence sequence analysis of amplified nucleic acid fragments, gene expression analysis, and the like. As a result, it was confirmed that the desired gene was activated in the transformed yeast obtained in Example 7.
  • bacto manufactured by Becton Dickinson
  • polypeptone manufactured by Nippon Pharmaceutical Co., Ltd.
  • yeast nitrogen base Without Amino Acid and Ammonium Sulfate manufactured by Becton Dickinson
  • 1% Ammonium Sulfate 0.4 mg / l Biotin, 100 mM phosphate Potassium (pH 6.0), 2% Glycerol
  • BMMY medium 1% yeast extract bacto (manufactured by Becton Dickinson), 2% polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.), 0.34% yeast nitrogen base Without Amino Acid and Ammonium Sulfate (manufactured by Becton Dickinson), 1% Ammonium Sulfate, 200 ⁇ l of preculture solution was subcultured in 0.4 mg / l Biotin, 100 mM potassium phosphate (pH 6.0), 2% Methanol), and this was cultured with shaking at 30 ° C., 170 rpm, 48 hours, and then centrifuged (12,000 rpm, 5 minutes). , 4 ° C) to collect the culture supernatant.
  • Protein L dissolved in (for Ray Biotech, tandem scFv226 antibody, brinattumomab antibody, minibody antibody) was added in 50 ⁇ l of each well and incubated overnight at 4 ° C. After incubation, the solution in the well was removed, blocked with 200 ⁇ l of immunoblock (manufactured by Sumitomo Dainippon Pharma), and allowed to stand at room temperature for 1 hour.
  • immunoblock manufactured by Sumitomo Dainippon Pharma
  • TMB-1 Component Microwell Peroxidase Substrate SureBlue manufactured by KPL
  • the absorbance at 450 nm was measured with a microplate reader (Envison; manufactured by PerkinElmer). The quantification of each small molecule antibody in the culture supernatant was performed using the calibration curve of each standard antibody.
  • Nucleic acid fragments with EcoRI recognition sequences added to both ends of the promoter-controlled noseoslisin resistance gene (SEQ ID NO: 142) and hyglomycin resistance gene (SEQ ID NO: 141) were added to Primer 88 (SEQ ID NO: 185) and Primer 94 (SEQ ID NO: 141), respectively. 192) and Primer 88 (SEQ ID NO: 185) and Primer 89 (SEQ ID NO: 186) were prepared by PCR and inserted into the EcoRI site of pUC-2 after EcoRI treatment to construct pUC2_NAT and pUC2_Hyg.
  • nucleic acid fragments of the first half and the second half of the MRP40 terminator were subjected to primer 116 (SEQ ID NO: 220), primer 117 for mutagenesis (SEQ ID NO: 221), and primer 118 for mutagenesis (SEQ ID NO: 221), respectively.
  • primer 116 SEQ ID NO: 220
  • primer 117 for mutagenesis SEQ ID NO: 221
  • primer 118 for mutagenesis SEQ ID NO: 221)
  • nucleic acid fragments of the first half and the second half of the ARG83 terminator were subjected to primer 120 (SEQ ID NO: 225), primer 121 for mutagenesis (SEQ ID NO: 226), and primer 122 for mutagenesis (SEQ ID NO: 226), respectively.
  • primer 120 SEQ ID NO: 225
  • primer 121 for mutagenesis SEQ ID NO: 226)
  • primer 122 for mutagenesis SEQ ID NO: 226)
  • pUC2_Hyg_TARG83 with a sequence into which the site (GGCGCGCC) was introduced.
  • Example 1 Preliminary study for preparation of Komagataera fafi gene overexpressing cell library As a gene overexpressing cell library that replaces the conventional cDNA library and genome library, the endogenous promoters of all yeast genes are highly expressed (GAPDH promoter).
  • GPDH promoter highly expressed
  • a plasmid library is prepared by treating pUC_del_Zeo_Pgap-EGFP-CYC1t prepared in (7) above with SpeI and XhoI, mixing with the above double-stranded OLS sequence, and connecting them using the In-fusion HD Cloning Kit.
  • the plasmid of the above library is linearly converted with the restriction enzyme BspQI, and the linear plasmid is inserted into the target position (each GeneX) on the genome of Komagataera fafi by homologous recombination by single crossover integration. ..
  • the base sequence from the start codon to the 91st and the base sequence from the 93rd to the 183rd of the Komagataera fafi gene KAR2 or PDI1 are extracted, and the 3'end sequence of the GAPDH promoter, the restriction enzyme SpeI recognition site, and the start codon to the 91st.
  • TGA termination codon
  • the KAR2 overexpression OLS sequence and the PDI1 overexpression OLS sequence linked in the order of the restriction enzyme recognition cleavage site XhoI and the CYC1 terminator derived from Saccharomyces cerevisiae were prepared (Fig. 2, SEQ ID NO: 200 (KAR2)). , SEQ ID NO: 201 (PDI1)).
  • Nucleic acid fragments were prepared after treating the overexpressing cell library preparation vector pUC_del_Zeo_Pgap-EGFP-CYC1t of (7) above with SpeI and XhoI, and the KAR2 overexpressing OLS sequence and PDI1 overexpressing OLS sequence prepared as described above were used. They were mixed and spliced together using the In-fusion HD Cloning Kit to prepare pUC_del_Zeo_Pgap-KAR2OLS-CYC1t and pUC_del_Zeo_Pgap-PDI1OLS-CYC1t.
  • the OLS sequence for overexpression of KAR2 or PDI1 was placed downstream of the GAPDH promoter derived from Komagataera fafi, and it was confirmed by the Sanger method that the nucleotide sequence was appropriate. After linearly converting these plasmids with the restriction enzyme BspQI, they were introduced into Komagataera fafi DNL4 deficient and histidine-requiring strains (Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018). It was inserted into the target site (KAR2 gene or PDI1 gene) by homologous recombination.
  • Primer sequence 102 (SEQ ID NO: 202) and primer sequence 103 (SEQ ID NO: 203) that sandwich the KAR2 gene on the genome, and primer sequence 104 (SEQ ID NO: 204) and primer sequence 105 (SEQ ID NO: 205) that sandwich the PDI1 gene. ) was designed, and the insertion of the above-mentioned plasmid into the target site of the Komagataera fafi genome was verified by the colony PCR method. The position of the band of the PCR product on agarose gel electrophoresis showed that each plasmid was inserted into the target site on the genome.
  • KAR2 or PDI1 gene of the inserted strain was overexpressed was evaluated by using the RT-qPCR method for the transcription amount of KAR2 and PDI1.
  • KAR2 overexpressing strain and PDI1 overexpressing strain using RNeasy kit (manufactured by Kiagen), reverse transcription using the reverse transcription kit (ReverTraAce qPCR RT Master Mix, manufactured by TOYOBO).
  • a photoreaction was performed, and the amount of KAR2 and PDI1 transcriptions of each strain was quantified using a quantitative PCR kit (KOD SYBR® qPCR Mix, manufactured by TOYOBO).
  • Primer sequence 106 (SEQ ID NO: 206) and primer sequence 107 (SEQ ID NO: 207) are used to quantify the amount of KAR2 transcription
  • primer sequence 108 (SEQ ID NO: 208) and primer sequence 109 (SEQ ID NO: 209) are used to quantify the amount of PDI1 transcription. It was used.
  • the ACT1 gene of Komagataera fafi was used, and primer sequence 110 (SEQ ID NO: 210) and primer sequence 111 (SEQ ID NO: 211) were used.
  • KAR2 overexpressing strains and PDI1 overexpressing strains as controls having the plasmids pUC_Arg4_Pgap_KAR2_T37552_Zeo and pUC_Arg4_Pgap_PDI1_T37552_Zeo in which the KAR2 and PDI1 genes and their terminator regions were introduced downstream of the GAPDH promoter were prepared and compared, respectively) were prepared and compared.
  • RT-qPCR it was shown that the KAR2 and PDI1 overexpressing strains prepared by the above method overexpressed their respective genes in the same manner as the respective control strains (Fig. 3). The above results indicate that it is possible to prepare a cell library overexpressing the Komagataera fafi gene by this method.
  • Example 2 Preparation of Komagataera fafi gene overexpressing cell library Based on the results of Example 1, the above OLS sequences were designed (Fig. 2) and prepared for all Komagataera fafi genes (5,001 genes) (Agilent). ⁇ Agilent). In order to convert the obtained total OLS sequence into double-stranded DNA, DNA is amplified by PCR using primers 112 (SEQ ID NO: 212) and primer 113 (SEQ ID NO: 213) complementary to both ends of the OLS sequence. I let you.
  • the double-stranded OLS sequence was ligated to the overexpressing cell library preparation vector pUC_del_Zeo_Pgap-EGFP-CYC1t described in (7) above using the In fusion method, and the Escherichia coli DH5 ⁇ strain was transformed with the obtained plasmid.
  • the obtained transformant colonies (4x10 5 pieces) were collected from a plurality of plates, and a plasmid was extracted using a Plasmamid Plus Midi kit (Qiagen) to prepare a plasmid library.
  • the 12 transformant colonies obtained here were cultured in LB medium, the plasmid was extracted, and the nucleotide sequence of the OLS sequence contained in the plasmid was confirmed by the Sanger method.
  • the obtained plasmid library was cleaved with the restriction enzyme BspQI, DNA was purified, and then introduced into a Komagataera fafi strain secreting small molecule antibodies (anti-lysothium scFv antibody, tandem scFv226, blinatumomab antibody) using an electroporation method. ..
  • Example 3 High-throughput screening using the amount of small molecule antibody secreted as an index
  • the transformant group (gene overexpressing cell library) obtained by the electroporation method in Example 2 was selected as a colony picker (PM-2, microtechnition).
  • a square plate of YPD agar medium supplemented with Zeocin was arranged in a 96-well format and colonized by culturing at 30 ° C., and this was used as a master plate. From the master plate, 96 strains were simultaneously inoculated using 96 pins into a deep well plate containing 0.5 mL of BMGY medium. After stirring and culturing at 30 ° C.
  • Example 4 Identification of useful factors The genes overexpressed in the obtained screening positive strain were identified by the following methods (Fig. 4) ((a) to (f) below are (a) to (a) to 4 in FIG. 4). Corresponds to (f)).
  • Gen Toru-kun manufactured by Takara Bio Inc.
  • Genomic DNA was fragmented by simultaneous treatment with multiple restriction enzymes (using any of the following (i) to (iii)).
  • i) After treating the genomic DNA with the 5'protruding end-type restriction enzymes EcoRI, SalI, NheI, BamHI and ClaI, the 5'protruding end of the fragmented DNA was blunt-ended with Klenow fragment.
  • Genomic DNA was treated with 5'protruding end-type restriction enzymes BamHI, BclI and BglII (the 5'protruding end has the same base sequence (GATC)).
  • Genomic DNA was treated with blunt-ended restriction enzymes BsaAI, BsaBI, BstZ17I, HpaI, PmlI, SnaBI and StuI.
  • Fragmented DNA was cyclized by self-ligation.
  • Escherichia coli DH5 ⁇ strain was transformed with cyclized DNA on LB agar medium supplemented with ampicillin and zeocin.
  • a plasmid was extracted from the transformant.
  • the overexpression vectors of 36 genes determined by the above method (36 expression vectors from pUC_Arg4_Pgap_EF1st-1_T37552_Zeo to pUC_Arg4_Pgap_EF3rd-9_T37552_Zeo) were applied to the Arg4 site of each small molecule antibody-producing strain used in Example 2 ( A strain introduced by the method described in 13) was prepared, and it was examined whether or not the amount of small molecule antibody secreted increased.
  • EF1st-1 has also been reported separately by the inventors. From the above, among the genes listed in Table 4, 32 types of factors excluding EF1st-1, EF2nd-8, EF3rd-2 and EF3rd-4 were newly discovered in this study. Next, it was investigated whether the useful factors obtained in a specific antibody-producing strain also affect the increase in the productivity of other antibodies.
  • Eighteen useful factor expression vectors (18 types from pUC_Arg4_Pgap_EF1st-1_T37552_Zeo to pUC_Arg4_Pgap_EF1st-18_T37552_Zeo) obtained from anti-lysothium scFv antibody-producing strains were introduced into tandem scFv226 antibody and blinatumomab antibody-producing strains. ..
  • the tandem scFv226 antibody-producing strain was 0.9 to 1.6 times (Table 5), and the blinatumomab antibody-producing strain was 1.0 to 2.3 times as much as the host strain.
  • the anti-lysothium scFv antibody-producing strain was 1.0 to 1.2 times (Table 7), and the blinatumomab antibody-producing strain was 1.0 to 1.8 times as much as the host strain. (Table 8).
  • Example 5 Development of a high-producing small molecule antibody strain by accumulating useful factors
  • the effect of promoting the production of small molecule antibodies by the useful factors obtained in the above screening is relatively low, 1.1 to 1.6 times (in tandem scFv226 production).
  • Met In order to increase the amount of small molecule antibody secreted, the accumulation of useful factors was examined. This time, a total of 8 of the top 4 factors (EF1st-1 to 4, Table 1) obtained from the anti-lysozyme scFv antibody strain and the top 4 factors (EF2nd-1 to 4, Table 2) obtained from the tandem scFv226 antibody strain. Factors were selected. By accumulating these, we aimed to prepare a high-producing strain of tandem scFv226 antibody.
  • each of the eight selected factors was introduced into the tandem scFv226-producing parent strain by the method described in (13) above, and the amount of tandem scFv226 antibody secreted was evaluated.
  • the high stock was used as the next-generation parent stock.
  • one gene-deficient strain (A strain) that produces tandem scFv226 antibody was used as the parent strain, and only the useful factor EF1st-1, which increased the amount of taFv226 antibody secreted most, was introduced into the ARG4 site, and the tandem scFv226 antibody was introduced. The amount of secretion was evaluated. As a result, a strain B in which the amount of tandem scFv226 antibody secreted was increased by about 1.5 times as compared with the strain A was obtained (Table 11). With strain B as the second-generation parent strain, each of the eight factors selected above was introduced into the URA3 site, and a strain in which each factor was overexpressed was prepared by the method described in (13) above.
  • the EF2nd-4 introduced strain (C strain) secreted the highest amount, and secreted about 1.2 times as much antibody as the parent strain B (Table 12). ).
  • the C strain was used as the third-generation parent strain, and the eight factors selected above were introduced into the GUT1 site, respectively, and a strain in which each factor was overexpressed was prepared by the method described in (13) above.
  • the EF1st-4 introduced strain (D strain) secreted the highest amount, and secreted about 1.2 times as much antibody as the parent strain C (Table 13). ).
  • each of the 8 factors selected above was introduced into the AOX1 promoter site, and a strain in which each factor was overexpressed was prepared by the method described in (13) above.
  • the EF2nd-1-introduced strain had the highest amount of secretion, and secreted about 1.3 times as much antibody as the parent strain D (Table 14).
  • This strain was designated as E strain.
  • E strain the E strain into which the four useful factors were introduced secreted tandem scFv226 antibody, which was about 2.9 times higher than that of the A strain before the introduction of the useful factors.
  • Example 6 Exchange of antibody expression cassette from antibody-producing strain If the antibody expression cassette of the antibody-producing strain can be inserted and removed, different antibodies can be produced in the tandem scFv226 high-producing strain prepared in Example 5. Therefore, we examined the exchange of gene expression cassettes using the Cre-loxP system. Specifically, when a loxP sequence (34 base length) is inserted at both ends of the genome transfer sequence of Komagataera fafi of the antibody expression cassette, the expression cassette introduced into Komagataera fafi has loxP sequences at both ends. (Fig. 6). Next, a plasmid pPAP_CP (Ito et al., FEMS Yeast Research, Vol. 18, No.
  • Cre recombinase gene regulated by a methanol-induced AOX1 promoter is introduced into an antibody-producing strain using a Zeo marker. ..
  • Cre recombinase expression at the leak level of the AOX1 promoter deletes the nucleotide sequence between loxPs.
  • the deletion of the antibody expression cassette can be evaluated by the colony PCR method using primers designed at both ends of the genome introduction site or the presence or absence of a drug marker in a drug assay.
  • a different antibody expression vector By introducing a different antibody expression vector into the same genome insertion site of the strain from which this antibody cassette has been deleted, expression from the tandem scFv226 antibody of the tandem scFv226 high-producing strain to a different antibody becomes possible (Fig. 6).
  • the protein expression cassette was exchanged using green fluorescent protein (GFP) and red fluorescent protein gene (RFP).
  • GFP green fluorescent protein
  • RFP red fluorescent protein gene
  • a gene encoding EGFP SEQ ID NO: 214
  • E2crimson SEQ ID NO: 215
  • pPGP_GFP As the GFP expression vector, pPGP_GFP (Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018) was used.
  • This vector is designed for GFP to be expressed under the control of the GAPDH promoter.
  • the RFP gene was prepared by PCR using synthetic DNA as a template and primer 114 (SEQ ID NO: 216) and primer 115 (SEQ ID NO: 217).
  • This nucleic acid fragment is prepared after SpeI and XhoI treatment, mixed with the nucleic acid fragment of the nucleotide sequence encoding RFP prepared by the above PCR, and spliced using the In-fusion HD Cloning Kit to obtain pPGP_RFP. It was constructed.
  • This vector is designed so that RFP is expressed under the control of the GAPDH promoter.
  • FIG. 7 shows the process of exchanging the reporter gene from the GFP-expressing strain to the RFP-expressing strain using the Komagataera fafi wild strain.
  • a strain into which a GFP expression cassette (pPGP_GFP fragment linearly converted at the EcoRV site in the CCA38473 terminator sequence) was introduced into the CCA38473 terminator site of Komagataera fafi (Fig. 7 (1)) was introduced into the above (13). It was produced by the method described in (Fig. 7 (2)). This strain was G418 resistant, and GFP fluorescence was confirmed by flow cytometry (FCM).
  • FCM flow cytometry
  • Example 7 Evaluation of antibody high-producing strains with different small molecule antibodies
  • loxP sequences were introduced at both ends of the sequence for introduction of the antibody expression vector into the CCA38473 terminator site. Therefore, loxP sequences were introduced at both ends of the antibody cassette introduced into the yeast genome (Fig. 8). Therefore, if Cre recombinase is expressed in yeast cells, the tandem scFv226 antibody expression cassette can be removed.
  • pPAP_CP Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018
  • E1, E2, E3 strains were prepared by the method described in (13) above.
  • the amount of each antibody secreted was evaluated by the ELISA method and SDS-PAGE, the amount of antibody secreted was 10 times or more in the anti-lysothium scFv antibody-producing strain and 3 times or more in the blinatumomab antibody-producing strain as compared with the host strain. confirmed.
  • the amount of antibody secreted was confirmed to be about twice that of the D3 strain derived from the D strain and about three times that of the E3 strain derived from the E strain (Fig. 9). From the above results, it was shown that the stocks of useful factors obtained by using the productivity of the tandem scFv226 antibody as an index have high productivity not only in the production of the tandem scFv226 antibody but also in different small molecule antibodies.
  • Example 8 Preparation of antibody production promoting protein overexpressing cell library for pair screening and screening of high-producing strains As shown in Example 5, the accumulation experiment conducted using eight kinds of useful factors showed low accumulation of useful factors. It was shown to be effective in promoting the production and secretion of molecular antibodies. Therefore, as a method for efficiently accumulating the obtained useful factors, a yeast library in which two kinds of useful factors were overexpressed at the same time was prepared, and a strain that secretes the most small molecule antibody from the library is shown in Example 3. We devised to find out using a high-throughput screening method (pair screening, FIG. 10).
  • a plasmid library consisting of a population of plasmids containing nucleotide sequences linked invertedly across the plate was prepared (Fig. 10, variety of combination types: 81).
  • the prepared plasmid library was introduced into the blinatumomab secretory strain prepared in (13) above to obtain about 2,400 transformants.
  • the transformants that appeared were screened using eight 96-well deep-well plates by the high-throughput screening method shown in Example 3.
  • the amount of blinatumomab secreted in the culture supernatant was evaluated by the ELISA method using the His tag as an index.
  • the amount of antibody secretion increased about 5 times as compared with the host strain (Table 15).
  • EF3rd-1 was introduced in both of them.
  • EF3rd-2 and EF3rd-5 were inserted.
  • the blinatumomab hypersecretory strain E2 strain (4 useful factor-introduced strains) obtained in the useful factor accumulation experiment in Example 7 had an antibody secretion amount of about 3.2 times that of the host strain. It was shown that the highly secreted strain (2 useful factor-introduced strain) obtained in 1) secreted a higher amount of blinatumomab antibody. Next, pair screening was performed in the second cycle using these two strains as parent strains. This time, a total of 10 EF1st-1 to 6 and EF2nd-1 to 4 were selected as useful factors for promoting blinatumomab secretion obtained in Example 2, and the combination was optimized.
  • a useful factor cassette (GAPDH promoter-useful factor) using two primer sets (primer 128 (SEQ ID NO: 233) and primer 129 (SEQ ID NO: 234), primer 130 (SEQ ID NO: 235) and primer 131 (SEQ ID NO: 236)). DNA was amplified by PCR using ORF and its terminator sequence). These two types of PCR products and pUC2_Hyg_TARG83 cleaved with BamHI and XhoI are mixed so that they have the same number of DNA molecules, and they are joined using the In-fusion HD Cloning Kit to obtain two useful factor expression cassettes.
  • a plasmid library consisting of a population of plasmids containing nucleotide sequences linked invertedly across the plate was prepared (Fig. 10, variety of combination types: 100).
  • the prepared plasmid library was introduced into the two types of blinatumomab hypersecretory strains shown above to obtain about 4,400 transformants. From the transformants that appeared, 9 96-well well plates (864 strains) were screened by the above high-throughput screening method.
  • the amount of secretion was 13 to 15 times higher than that of the host strain. Was shown (Table 15).
  • EF1st-1 was introduced into all the strains (Table 15).
  • the other useful factors were EF2nd-4 (same gene as EF3rd-2) (2nd_11-7 strain and 2nd_11-8 strain) and EF1st-3 (2nd_12-2 strain and 2nd_12-7 strain) (Table). 15).
  • the blinatumomab antibody hypersecreting strain contained useful factors in a combination different from the optimal combination in the tandem scFv226-producing strain shown in Example 7. It is considered that there is an optimal combination of useful factors depending on the modality and amino acid sequence of the small molecule antibody.
  • Example 9 Pair screening with different small molecule antibodies
  • the pair screening method was repeated twice to obtain a high-producing strain of blinatumomab antibody.
  • the useful factors selected as the optimal combination in the blinatumomab hypersecretory strain were EF1st-1, EF1st-3, EF2nd-4, EF3rd-1 and EF3rd-5.
  • a pair screening method using the blinatumomab antibody shown in Example 8 was carried out to try to identify useful factors to be selected.
  • Small molecule antibody (tandem scFv226) secretory strain prepared in (13) above using a plasmid library consisting of 9 useful factors (EF3rd-1-9) prepared in the first cycle of pair screening in Example 8. Introduced into, 1,200 transformants were obtained. From the transformants that appeared, screening for 4 96-well deep-well plates (384 strains) was performed by the high-throughput screening method shown in Example 3. The amount of taFv226 secreted in the culture supernatant was evaluated by the ELISA method shown in (15) above.
  • Example 8 In the first generation pair screening in Example 8, the optimal combination of useful factors was EF3rd-1 and EF3rd-2 (EF2nd-4), EF3rd-1 and EF3rd-5, and the tandem scFv226 secretion of this example. It was different from EF3rd-2 (EF2nd-4) and EF3rd-3 obtained by screening using strains. That is, from this result, it was shown that the optimum combination of useful factors for the secretion of each small molecule antibody is different.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Mycology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention fournit un procédé de préparation de cellules génétiquement modifiées ayant une expression génique endogène améliorée, une cellule génétiquement modifiée préparée par ledit procédé, et un procédé de criblage d'un gène endogène qui améliore la production d'une protéine cible à l'aide d'une banque de cellules surexprimant un gène endogène.
PCT/JP2021/011093 2020-03-19 2021-03-18 Procédé de préparation de cellules génétiquement modifiées ayant une expression génique endogène améliorée WO2021187560A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2022508434A JPWO2021187560A1 (fr) 2020-03-19 2021-03-18

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2020050192 2020-03-19
JP2020-050192 2020-03-19

Publications (1)

Publication Number Publication Date
WO2021187560A1 true WO2021187560A1 (fr) 2021-09-23

Family

ID=77771026

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2021/011093 WO2021187560A1 (fr) 2020-03-19 2021-03-18 Procédé de préparation de cellules génétiquement modifiées ayant une expression génique endogène améliorée

Country Status (2)

Country Link
JP (1) JPWO2021187560A1 (fr)
WO (1) WO2021187560A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012213376A (ja) * 2011-03-31 2012-11-08 Ajinomoto Co Inc γ−グルタミル化合物を含有する酵母エキスの製造方法及び当該方法に用いられる酵母
JP2019507748A (ja) * 2016-02-12 2019-03-22 アブリンクス エン．ヴェー． 免疫グロブリン単一可変ドメインの生成方法
WO2019187911A1 (fr) * 2018-03-26 2019-10-03 国立大学法人神戸大学 Nouvelle cellule et procédé de production d'une protéine d'intérêt l'utilisant

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012213376A (ja) * 2011-03-31 2012-11-08 Ajinomoto Co Inc γ−グルタミル化合物を含有する酵母エキスの製造方法及び当該方法に用いられる酵母
JP2019507748A (ja) * 2016-02-12 2019-03-22 アブリンクス エン．ヴェー． 免疫グロブリン単一可変ドメインの生成方法
WO2019187911A1 (fr) * 2018-03-26 2019-10-03 国立大学法人神戸大学 Nouvelle cellule et procédé de production d'une protéine d'intérêt l'utilisant

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YASUYUKI OTAKE ET AL.: "Glutathione koseisan kobo no ikushu", BIOSCIENCE TO INDUSTRY, XX, XX, vol. 50, no. 10, 1 January 1992 (1992-01-01), XX , pages 989 - 994, XP002970454 *

Also Published As

Publication number Publication date
JPWO2021187560A1 (fr) 2021-09-23

Similar Documents

Publication Publication Date Title
US11976284B2 (en) Promoter variants for protein production
Çelik et al. Production of recombinant proteins by yeast cells
JP5932766B2 (ja) Ｖｐｓ遺伝子が破壊されている酵母を用いる異種タンパク質の製造方法
Fowler et al. Gene expression systems for filamentous fungi
US11485979B2 (en) Cell and method for producing target protein using same
WO2021187560A1 (fr) Procédé de préparation de cellules génétiquement modifiées ayant une expression génique endogène améliorée
EP2489724A1 (fr) Levure hansenula polymorpha apte à produire un anticorps, procédé de production d'anticorps la faisant intervenir, et anticorps produit à partir de celle-ci
JP6943841B2 (ja) メタノール資化性酵母由来新規タンパク質及びそれを用いた目的タンパク質の製造方法
JP7239205B2 (ja) 新規宿主細胞及びそれを用いた目的タンパク質の製造方法
Felber et al. Strains and molecular tools for recombinant protein production in Pichia pastoris
AU2016329244B2 (en) Novel episomal plasmid vectors
JP2016146789A (ja) 新規オガタエア属酵母及びそれを用いた異種タンパク質の製造方法。
JP7012663B2 (ja) 新規宿主細胞及びそれを用いた目的タンパク質の製造方法
JP6387346B2 (ja) 新規ポリペプチド及びその用途
JP6387345B2 (ja) 新規ポリペプチド及びその用途
WO2023104896A1 (fr) Production améliorée de protéines sécrétées dans des cellules fongiques
JP2020048536A (ja) 新規細胞及びそれを用いた目的タンパク質の製造方法
Rudolph et al. Expression of Recombinant Proteins in Yeast
do Espírito Santo Pesquisa de Estirpes de Saccharomyces Cerevisiae Para expressão de proteína Recombinante

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21772466

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2022508434

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21772466

Country of ref document: EP

Kind code of ref document: A1