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1. WO2022089573 - RECOMBINANT VECTOR AND EXPRESSION PURIFICATION METHOD FOR HEAT-LABILE UNG FUSION PROTEIN

Publication Number WO/2022/089573
Publication Date 05.05.2022
International Application No. PCT/CN2021/127313
International Filing Date 29.10.2021
IPC
C12N 15/70 2006.1
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
70Vectors or expression systems specially adapted for E. coli
C12N 9/24 2006.1
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
9Enzymes, e.g. ligases (6.); Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating, or purifying enzymes
14Hydrolases (3.)
24acting on glycosyl compounds (3.2)
C12N 15/56 2006.1
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
11DNA or RNA fragments; Modified forms thereof
52Genes encoding for enzymes or proenzymes
55Hydrolases (3)
56acting on glycosyl compounds (3.2), e.g. amylase, galactosidase, lysozyme
CPC
C07K 2319/21
CCHEMISTRY; METALLURGY
07ORGANIC CHEMISTRY
KPEPTIDES
2319Fusion polypeptide
20containing a tag with affinity for a non-protein ligand
21containing a His-tag
C07K 2319/95
CCHEMISTRY; METALLURGY
07ORGANIC CHEMISTRY
KPEPTIDES
2319Fusion polypeptide
95containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
C12N 15/70
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
70Vectors or expression systems specially adapted for E. coli
C12N 9/2497
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
9Enzymes; Proenzymes; Compositions thereof
14Hydrolases (3)
24acting on glycosyl compounds (3.2)
2497hydrolysing N- glycosyl compounds (3.2.2)
Applicants
  • 厦门大学 XIAMEN UNIVERSITY [CN]/[CN]
  • 厦门致善生物科技股份有限公司 XIAMEN ZEESAN BIOTECH CO., LTD. [CN]/[CN]
Inventors
  • 张永有 ZHANG, Yongyou
  • 王丹 WANG, Dan
  • 宋娜杰 SONG, Najie
Agents
  • 厦门市首创君合专利事务所有限公司 SHOUCHUANG JUNHE PATENT AGENT CO., LTD. XIAMEN
Priority Data
202011191136.730.10.2020CN
Publication Language Chinese (zh)
Filing Language Chinese (ZH)
Designated States
Title
(EN) RECOMBINANT VECTOR AND EXPRESSION PURIFICATION METHOD FOR HEAT-LABILE UNG FUSION PROTEIN
(FR) VECTEUR RECOMBINÉ ET PROCÉDÉ DE PURIFICATION D'EXPRESSION POUR UNE PROTÉINE DE FUSION UNG LABILE À LA CHALEUR
(ZH) 一种不耐热UNG融合蛋白的重组载体及表达纯化方法
Abstract
(EN) Provided are a recombinant vector and an expression purification method for a heat-labile UNG fusion protein. The method comprises: cloning a gene sequence of Cod UNG to a pCold-sumo plasmid to construct a recombinant plasmid pCold-sumo-Cod UNG, transforming the recombinant plasmid into an Escherichia coli BL21 (DE3) competent cell, simultaneously transforming a plasmid pG-Tf2 which expresses chaperone into the competent cell, and carrying out low-temperature-induced expression to obtain a soluble sumo-Cod UNG fusion protein. According to the method, the problem of insoluble Cod UNG expression products is overcome.
(FR) L'invention concerne un vecteur recombiné et un procédé de purification d'expression pour une protéine de fusion UNG labile à la chaleur. Le procédé comprend les étapes suivantes : clonage d'une séquence de gène de Cod UNG dans un plasmide pCold-sumo pour construire un plasmide recombiné pCold-sumo-Cod UNG; transformation du plasmide recombiné dans une cellule compétente Escherichia coli BL21 (DE3); transformation simultanée d'un plasmide pG-Tf2 exprimant une chaperonne dans la cellule compétente; et réalisation d'une expression induite à basse température pour obtenir une protéine de fusion soluble sumo-Cod UNG. Le procédé permet de surmonter le problème des produits d'expression insolubles de Cod UNG.
(ZH) 提供了一种不耐热UNG融合蛋白的重组载体及表达纯化方法,包括将Cod UNG的基因序列克隆到pCold-sumo质粒构建重组质粒pCold-sumo-Cod UNG,转化大肠杆菌BL21(DE3)感受态,同时转化表达分子伴侣质粒pG-Tf2,经低温诱导表达得到可溶性的sumo-Cod UNG融合蛋白,解决了Cod UNG表达产物不溶的问题。
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