WO2009058956A1 - Signal sequences and co-expressed chaperones for improving protein production in a host cell - Google Patents

Signal sequences and co-expressed chaperones for improving protein production in a host cell Download PDF

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Publication number
WO2009058956A1
WO2009058956A1 PCT/US2008/081721 US2008081721W WO2009058956A1 WO 2009058956 A1 WO2009058956 A1 WO 2009058956A1 US 2008081721 W US2008081721 W US 2008081721W WO 2009058956 A1 WO2009058956 A1 WO 2009058956A1
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Prior art keywords
sequence
protein
laccase
expression
host cell
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Ceased
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PCT/US2008/081721
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English (en)
French (fr)
Inventor
Kai Bao
Huaming Wang
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Danisco US Inc
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Danisco US Inc
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Priority to CA2704548A priority Critical patent/CA2704548A1/en
Priority to BRPI0818273-6A2A priority patent/BRPI0818273A2/pt
Priority to EP08844940A priority patent/EP2203556A1/en
Priority to CN200880114429A priority patent/CN101842479A/zh
Priority to AU2008318644A priority patent/AU2008318644B2/en
Priority to JP2010532232A priority patent/JP5252746B2/ja
Priority to MX2010004325A priority patent/MX2010004325A/es
Publication of WO2009058956A1 publication Critical patent/WO2009058956A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C12N9/0061—Laccase (1.10.3.2)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
    • C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
    • C12Y110/03002—Laccase (1.10.3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • glucoamylase refers to the amyloglucosidase class of enzymes (e.g., EC.3.2.1.3, glucoamylase, 1, 4-alpha-D-glucan glucohydrolase). These are exo-acting enzymes, which release glucosyl residues from the non-reducing ends of amylose and amylopectin molecules. The enzyme also hydrolyzes alpha- 1, 6 and alpha -1,3 linkages although at much slower rate than alpha- 1, 4 linkages.
  • expression vector means a DNA construct including a DNA sequence which is operably linked to a suitable control sequence capable of affecting the expression of the DNA in a suitable host.
  • filamentous fungi refers to all filamentous forms of the subdivision Eumycotina, as known in the art. These fungi are characterized by a vegetative mycelium with a cell wall composed of chitin, cellulose, and other complex polysaccharides.
  • the filamentous fungi of the present invention are morphologically, physiologically, and genetically distinct from yeasts. Vegetative growth by filamentous fungi is by hyphal elongation and carbon catabolism is obligatory aerobic.
  • the specific signal peptide used in the present invention is not critical, as long as the signal peptide is operable in the host.
  • An "operable signal peptide” is provided when the signal peptide increases secretion of a protein when operably linked to the protein in a host cell.
  • the signal peptide is obtained from a strongly secreted protein and/or is a strong signal peptide.
  • a "strong signal peptide” results when the natural protein is strongly secreted by its natural host.
  • the signal peptide is obtained from an organism within the same phylum as the host cell. Indeed, in some embodiments, this is advantageous.
  • the signal sequence begins at base pair 210 and ends at base pair 260 (SEQ ID NO: 11).
  • the catalytic core begins at base pair 261 through base pair 1698 (SEQ ID NO: 12), including intron 1 (from base pair 671 to 737) and intron 2 (from base pair 1435 to 1497).
  • the linker sequence begins at base pair 1699 and ends at base pair 1770 (SEQ ID NO: 13).
  • the cellulose binding domain begins at base pair 1771 through base pair 1878.
  • the sequence and domain information for CBHI can be found via the expasy organization website and is designated uniprot/P62694. CBHI homologs have been identified in a number of other Trichoderma species as well as other filamentous fungi and find use in the present invention as appropriate.
  • the derivative proteins differ by as few as about 1 to about 10 amino acid residues, such as about 6 to about 10, as few as about 5, as few as about 4, about 3, about 2, or even 1 amino acid residue, compared to the "parent" protein sequence.
  • Table 1 provides exemplary conservative amino acid substitutions recognized in the art.
  • substitution involves one or more non-conservative amino acid substitutions, deletions, or insertions that do not abolish the signal peptide activity.
  • Homology of DNA sequences is determined by the degree of identity between two DNA sequences. Homology or “percent identity” is often determined for polypeptide sequences and/or nucleotides sequences using computer programs. Methods for performing sequence alignment and determining sequence identity are well-known to the skilled artisan, may be performed without undue experimentation, and calculations of identity values are obtainable with definiteness. A number of algorithms are available and known to those of skill in the art, for aligning sequences and determining sequence identity.
  • promoters include cbhl, cbh2, egll, egl2, egl3, egl4, egl5, xynl, and xyn2, repressible acid phosphatase gene (phoA) promoter of P. chrysogenum ⁇ See, Graessle et al, Appl. Environ.
  • phoA repressible acid phosphatase gene
  • the present invention be limited to any particular type of protein. Indeed, it is intended that the present invention encompass any protein of interest.
  • desired proteins include, but are not limited to glucoamylases, alpha amylases, granular starch hydrolyzing enzymes, cellulases, lipases, xylanases, cutinases, hemicellulases, proteases, oxidases, laccases and combinations thereof.
  • Desired proteins of the present invention are produced by culturing cells transformed with a vector such as an expression vector containing genes whose secretion is enhanced by the NSP24 or CB ⁇ 1 signal peptide sequence, foldases, chaperonins, and/or chaperones.
  • the present invention is particularly useful for enhancing the intracellular and/or extracellular production of proteins.
  • optimal conditions for the production of the proteins will vary with the choice of the host cell and protein to be expressed. Such conditions are easily determined by those of skill in the art.
  • the precipitated proteins are then solubilized and in some embodiments, are purified by any suitable method, including chromatographic procedures (e.g., ion exchange chromatography, gel filtration chromatography, affinity chromatography, and other art-recognized procedures).
  • chromatographic procedures e.g., ion exchange chromatography, gel filtration chromatography, affinity chromatography, and other art-recognized procedures.
  • the culture medium of the transformants obtained and cultivated as described in Example 8 was separated from mycelium by centrifugation (16000 xg, 10 min) and ABTS activity from the supernatants were analyzed. The results are shown in Figure 10.
  • Table 3 provides the strains described in Figure 10.
  • Figure 10 illustrates the improvement of laccase production by fusion of the gene encoding C, unicolor laccase to the full-length Trichoderma glucoamylase. The results showed that expression of laccase improved 24-29% when fused to the Trichoderma glucoamylase, than fused to CBHl .
  • the CBHl signal sequence plasmid ⁇ i.e., operably linked to laccase was co- transformed with a variety of T. reesei chaperone plasmids ⁇ bipl, lhsl , pdil , ppil , ppi2 ', tigl, prpl, and erol), either alone or in combination.
  • the cultures were grown in shake flasks as known in the art and laccase expression analyzed using the ABTS assay.
  • the clones were analyzed in triplicate.
  • the data provided in Table 4 show that adding more than one chaperone did not increase expression of laccase above that of bipl alone.
  • the data in Table 4 show three independent spore-purified samples (or clones) from the same strain.

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  • Life Sciences & Earth Sciences (AREA)
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  • Engineering & Computer Science (AREA)
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  • Wood Science & Technology (AREA)
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  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
PCT/US2008/081721 2007-11-01 2008-10-30 Signal sequences and co-expressed chaperones for improving protein production in a host cell Ceased WO2009058956A1 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
CA2704548A CA2704548A1 (en) 2007-11-01 2008-10-30 Signal sequences and co-expressed chaperones for improving protein production in a host cell
BRPI0818273-6A2A BRPI0818273A2 (pt) 2007-11-01 2008-10-30 Sequências sinalizadoras e chaperonas coexpressas para aprimoramento de produção de proteína em uma célula hospedeira.
EP08844940A EP2203556A1 (en) 2007-11-01 2008-10-30 Signal sequences and co-expressed chaperones for improving protein production in a host cell
CN200880114429A CN101842479A (zh) 2007-11-01 2008-10-30 用于改良宿主细胞内蛋白质生产的信号序列和共表达的分子伴侣
AU2008318644A AU2008318644B2 (en) 2007-11-01 2008-10-30 Signal sequences and co-expressed chaperones for improving protein production in a host cell
JP2010532232A JP5252746B2 (ja) 2007-11-01 2008-10-30 宿主細胞におけるタンパク質生産を改善するためのシグナル配列及び共発現したシャペロン
MX2010004325A MX2010004325A (es) 2007-11-01 2008-10-30 Secuencias de señal y chaperones co-expresados para mejorar produccion de proteinas en una celula hospedera.

Applications Claiming Priority (2)

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US98443007P 2007-11-01 2007-11-01
US60/984,430 2007-11-01

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EP (1) EP2203556A1 (enExample)
JP (3) JP5252746B2 (enExample)
CN (1) CN101842479A (enExample)
AU (1) AU2008318644B2 (enExample)
BR (1) BRPI0818273A2 (enExample)
CA (1) CA2704548A1 (enExample)
CO (1) CO6210760A2 (enExample)
MX (1) MX2010004325A (enExample)
WO (1) WO2009058956A1 (enExample)

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WO2010139858A1 (en) * 2009-06-02 2010-12-09 Oulun Yliopisto Method for producing natively folded proteins in a prokaryotic host
JP2012235767A (ja) * 2011-04-27 2012-12-06 Toyota Motor Corp 変異トリコデルマ属微生物及びこれを用いたタンパク質の製造方法
WO2013102674A3 (en) * 2012-01-05 2013-11-14 Novartis International Pharmaceutical Ltd. Protease deficient filamentous fungal cells and methods of use thereof
US20140271720A1 (en) * 2013-03-12 2014-09-18 Wisconsin Alumni Research Foundation Method of Treating Fungal Infection
CN112175977A (zh) * 2020-10-30 2021-01-05 华中农业大学 一种米曲霉角蛋白酶基因及其表达载体与应用
CN112409464A (zh) * 2020-11-23 2021-02-26 江南大学 提高枯草芽孢杆菌重组蛋白胞外生产水平的信号肽突变体及应用

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CN103189564A (zh) 2010-10-18 2013-07-03 丹尼斯科美国公司 使用漆酶体系对染色织物进行局部颜色修改
EP2694647A1 (en) 2011-04-06 2014-02-12 Danisco US Inc. Laccase variants having increased expression and/or activity
WO2013111754A1 (ja) * 2012-01-23 2013-08-01 旭硝子株式会社 発現ベクターおよび蛋白質の製造方法
DK2852610T3 (en) 2012-05-23 2018-09-03 Glykos Finland Oy PRODUCTION OF FUCOSYLED GLYCOPROTEIN
MX2016000306A (es) 2013-07-10 2016-08-08 Novartis Ag Celulas de hongos filamentosos deficientes en multiples proteasas y métodos de uso de las mismas.
CN104694560A (zh) * 2013-12-04 2015-06-10 中国科学院天津工业生物技术研究所 一种源于里氏木霉的二硫键异构酶基因Trpdi2及其用途
WO2015158808A2 (en) 2014-04-17 2015-10-22 Boehringer Ingelheim Rcv Gmbh & Co Kg Recombinant host cell engineered to overexpress helper proteins
SG11201608080RA (en) 2014-04-17 2016-10-28 Boehringer Ingelheim Rcv Gmbh Recombinant host cell for expressing proteins of interest
SG11201700446XA (en) 2014-07-21 2017-02-27 Glykos Finland Oy Production of glycoproteins with mammalian-like n-glycans in filamentous fungi
WO2016025825A1 (en) * 2014-08-15 2016-02-18 Danisco Us Inc Compositions and methods for improved protein production
US10526609B2 (en) * 2016-08-01 2020-01-07 Aduro Biotech, Inc. Protein expression enhancer sequences and use thereof
AU2019294515B2 (en) * 2018-06-27 2025-10-09 Boehringer Ingelheim Rcv Gmbh & Co Kg Means and methods for increased protein expression by use of transcription factors
KR102138272B1 (ko) * 2018-09-19 2020-07-28 주식회사 바이오앱 BiP 단편을 포함하는 재조합 벡터 및 상기 벡터를 이용한 재조합 단백질의 제조 방법
CN120399007A (zh) * 2019-12-02 2025-08-01 株式会社Lg化学 Cho细胞衍生的蛋白分泌因子和包含它的表达载体
CN112501141A (zh) * 2020-10-22 2021-03-16 重庆中元汇吉生物技术有限公司 一种增加人甲状腺过氧化物酶产量的试剂和表达方法
CN112391402B (zh) * 2020-11-17 2023-03-28 华中科技大学 一种提高解脂耶氏酵母中目的蛋白表达水平的方法
US20240376512A1 (en) * 2021-09-09 2024-11-14 Dupont Nutrition Biosciences Aps Over expression of foldases and chaperones improves protein production
CN113736817B (zh) * 2021-10-08 2023-02-03 枣庄市杰诺生物酶有限公司 一种毕赤酵母中提高碱性脂肪酶分泌效率及酶活的方法
CN117903295B (zh) * 2024-03-19 2024-05-31 北京国科星联科技有限公司 一种用于分泌表达乳铁蛋白的马克斯克鲁维酵母及其构建方法与应用
CN117903294B (zh) * 2024-03-19 2024-06-14 北京国科星联科技有限公司 一种发酵生产乳铁蛋白的马克斯克鲁维酵母及其构建方法与应用

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JP5252746B2 (ja) 2013-07-31
CN101842479A (zh) 2010-09-22
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JP2015027308A (ja) 2015-02-12
US20090221030A1 (en) 2009-09-03
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EP2203556A1 (en) 2010-07-07
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