WO2017075863A1 - Vecteur d'expression de protéine hybride de protéine du type chaperon - Google Patents

Vecteur d'expression de protéine hybride de protéine du type chaperon Download PDF

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WO2017075863A1
WO2017075863A1 PCT/CN2015/097269 CN2015097269W WO2017075863A1 WO 2017075863 A1 WO2017075863 A1 WO 2017075863A1 CN 2015097269 W CN2015097269 W CN 2015097269W WO 2017075863 A1 WO2017075863 A1 WO 2017075863A1
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protein
expression vector
fusion protein
free fatty
fatty acid
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PCT/CN2015/097269
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English (en)
Chinese (zh)
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李万波
陕婧婧
卢婵
管春爱
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盘古基因生物工程(南京)股份有限公司
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Publication of WO2017075863A1 publication Critical patent/WO2017075863A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • the invention relates to a fusion protein expression technology, in particular to a fusion protein expression vector of a chaperone protein, belonging to the field of genetic engineering and protein expression engineering.
  • a protein depends on the natural conformation of the protein. It is generally believed that the tertiary and quaternary structure of the protein molecule is entirely determined by the amino acid sequence of the polypeptide.
  • Molecular chaperones are a class of evolutionarily very conserved proteins that bind non-specifically to polypeptide chains that differ in structure, size, location, and final function. They catalyze the formation of specific conformations of proteins and participate in the folding, assembly, and transport of proteins in vivo. .
  • HSP heat shock protein
  • molecular chaperones are a class of proteins that are related to each other and are capable of binding and stabilizing the unstable conformation of another protein. Their function is to help other peptides. Structured substances undergo correct non-covalent assembly in vivo, controlled binding and release, promotion of folding of nascent polypeptides, assembly or degradation of multimers, and transmembrane transport of organelle proteins, and are not assembled proteins. A component of its normal biological function [3] .
  • Proteins also known as accessory proteins that aid in the folding of nascent peptides are known to have at least three broad categories: one is a universal molecular chaperone that helps correct folding, prevents and corrects incorrect folding.
  • the other type is a molecular chaperone with enzyme activity, also known as a folding enzyme.
  • PDI protein disulfide isomerase
  • PPI peptidylprodylcis-trans isomerase
  • Ellis made it clear that PDI is not a molecular chaperone [3] , and it has been shown that these two isomerases are both enzymes and molecular chaperones [4] .
  • the third category is intramolecular chaperones.
  • leader peptide proteins that are synthesized in the precursor form of the leader peptide (Pro peptide) must be folded and matured to have the presence of a Pro peptide, and do not fully comply with the Anfinsen rule.
  • leader peptides are called intramolecular chaperones (IMCs).
  • Dong Xiaoyan et al. used the immobilized molecular chaperone GroE to study its renaturation effect on denatured lysozyme and solved the problem of molecular chaperone reuse.
  • Teshima et al. assisted the folding renaturation of amylase, carbonic anhydrase, and DNase in vitro using immobilized molecular chaperones. It has also been reported that renaturation of target proteins by "small molecular chaperones" can effectively promote the renaturation of cyclophilin A, rhodanese and bacillus RNase.
  • GST Bacterial glutathione S-transferase
  • NEB uses maltose binding protein to construct pMAL series prokaryotic expression vector, which can also promote the soluble expression of partial fusion protein, has chaperone-like protein characteristics, and further utilizes the affinity of MBP for maltose to achieve amylose.
  • the (Amylose) column was affinity purified for the fusion protein.
  • DsbA Disulfide bond formation protein A
  • Prokaryotic expression vectors can contribute to increased solubility of the expressed exogenous protein.
  • DsbA belongs to a chaperone protein.
  • Takara Bio INC. constructed and sold a series of co-transfected prokaryotic expression vectors containing cpn60, in an attempt Improve the renaturation of the expressed rim protein and achieved good results.
  • Previous studies have shown that GroEL can significantly improve the refolding efficiency of genetically engineered protein scorpion Cn5, cyclophilin A and ⁇ 3-glycerol phosphate synthase in vitro.
  • Small ubiquitin-like modified proteins are widely found in eukaryotes and are a class of small-molecule polypeptides involved in post-translational modification of proteins. They have good self-folding properties during E. coli expression and also induce some fusions downstream. The folding action of proteins is classified as a chaperon-like protein. Such prokaryotic expression vectors containing SUMO have been widely used in foreign countries.
  • the optimized human free fatty acid binding protein coding sequence is inserted into the fusion protein expression vector as an upstream protein.
  • a fusion protein expression vector for a chaperon-like protein comprising a nucleic acid sequence encoding a human free fatty acid binding protein upstream of a cloning region thereof.
  • the fusion protein expression vector downstream of the human free fatty acid binding protein, preferably comprises a flexible linker region and a polyclonal region for insertion of the protein of interest.
  • nucleic acid sequence encoding the human free fatty acid binding protein is a codon-optimized coding sequence
  • the homology of the encoded human free fatty acid binding protein and the homology of the human natural free fatty acid binding protein is equal to or greater than 85 %.
  • the nucleic acid sequence encoding the human free fatty acid binding protein is further preferably as shown in SEQ ID NO: 10.
  • the flexible linker region and the polyclonal region sequence located downstream of the nucleic acid sequence encoding the human free fatty acid binding protein in the expression vector are preferably as set forth in SEQ ID NO: 13.
  • the upstream of the nucleic acid sequence encoding the human free fatty acid binding protein in the expression vector preferably further comprises a sequence encoding a tag for isolating and purifying the fusion protein, and further preferably a sequence encoding a histidine tag.
  • a method for expressing a fusion protein of a chaperone-like protein which comprises inserting a coding sequence of a target protein into a polyclonal region downstream of the flexible linker region of the expression vector according to any one of claims 3 to 8 to obtain a recombinant expression vector containing the coding sequence of the fusion protein.
  • the vector is transfected into a host cell, and the host cell is cultured to express the fusion protein.
  • the recombinant expression vector constructed by the present invention is capable of promoting or inducing folding of a fused downstream protein, having the properties of a protein chaperone-like protein;
  • the FABP protein is human-derived, and theoretically, it has no immunogenicity to the human body, and is suitable for solving the problem of target protein inclusion bodies in the pharmaceutical industry, and can retain the fusion protein together.
  • FIG. 1 Schematic diagram of hFABP fusion protein
  • a method for constructing a fusion protein expression vector of a chaperon-like protein comprising the steps of:
  • Step 1 Artificial synthesis and optimization of the coding DNA of hFABP.
  • hFABP6 is taken as an example (SEQ ID NO: 6):
  • the hFBP6 amino acid sequence was reverse translated into a DNA coding sequence, and the sequence was obtained by codon preference and ribosome binding region sequence optimization (SEQ ID NO: 10), but not limited to this sequence, and the translated protein amino acid sequence was the same.
  • the source is equal to or greater than 85%.
  • PCR Polymerase chain reaction
  • the hFABP6 coding sequence (SEQ ID NO: 29) synthesized in the first step, comprising a 5'-6xHisTag, a 3'-flexible linker sequence (Linker) and a polyclonal region, which are digested with restriction endonucleases,
  • the agarose gel was purified to obtain an insert of a viscous linker with Nco I and Xho I, respectively.
  • the expression vector pET28a is prepared, but is not limited to the expression vector:
  • the agarose gel electrophoresis was used to separate and purify the linearized pET28a vector.
  • the E. coli competent strain DH5 ⁇ was transformed, and the culture dish containing kanamycin antibiotic was cultured overnight, then single colonies were picked, positive clones were identified by PCR, and DNA plasmids were routinely prepared.
  • the positive clone plasmid was sent to DNA sequence analysis, and the clone of the correct sequence was selected and retained for expression test.
  • the pET28a-hFABP6 vector DNA plasmid obtained in the second step was transformed into E. coli BL21 (DE3) competent cells to obtain Kan-resistant colonies.
  • hFABP6 including N-HisTag, flexible linker, and polyclonal region, totaling 158 aa, molecular weight 17.2 kDa.
  • hFABP6 accounts for 20% of total bacterial proteins and is soluble in expression.
  • a DNA sequence encoding a hEGF mature peptide (SEQ ID NO: 32);
  • PCR method introduces Kpn I (5'-end) and Xho I (3'-end) double-enclosure cloning sites;
  • Upstream primer 5'-TTTTGGTACCAACTCTGACTCTGAATGCC-3' (SEQ ID NO: 33), downstream primer: 5'-TTTTCTCGAGTTAACGCAGCTCCCACCATTTGAG-3', (SEQ ID NO: 34)
  • the T4 DNA ligase is ligated to the pET28a-hFABP6 vector fragment and the hEGF insert DNA, and the recombinant expression vector comprises the histidine tag coding sequence represented by SEQ ID NO: 35 - hFABP6 protein coding sequence - flexible linker region - hEGF Insertion DNA, the corresponding amino acid sequence of which is shown in SEQ ID NO: 36;
  • the recombinant expression vector obtained in the previous step was directly transformed into E. coli BL21 (DE3) competent cells, and single colony PCR was used for identification.
  • the PCR primers used were pET28a sequencing primers T7-primer (SEQ ID NO: 37) and T7-terminator. . (SEQ ID NO: 38)
  • PCR method introduces Nco I (5'-end) and Xho I (3'-end) double restriction enzyme cloning site and initiation codon ATP; upstream primer: 5'-TTTTCCATGAACTCTGACTCTGAATGCC-3' (SEQ ID NO: 40), downstream primer: 5'-TTTTCTCGAGTTAACGCAGCTCCCACCATTTGAG-3' (SEQ ID NO: 34)
  • T4 DNA ligase is ligated to the pET28a DNA fragment and the hEGF DNA fragment;
  • IPTG induces protein expression
  • the upstream protein hFABP6 has a protein chaperone-like effect that promotes hEGF folding.

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  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un vecteur d'expression de protéine hybride d'une protéine du type chaperon. La région de clonage en amont du vecteur d'expression comprend une séquence nucléotidique codant une protéine de liaison aux acides gras libres d'origine humaine. La séquence nucléotidique en aval comprend de préférence une région articulée souple et une région de multi-clonage pour l'insertion d'un acide nucléique codant la protéine cible.
PCT/CN2015/097269 2015-11-05 2015-12-14 Vecteur d'expression de protéine hybride de protéine du type chaperon WO2017075863A1 (fr)

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CN201510747174.9 2015-11-05
CN201510747174.9A CN105296517B (zh) 2015-11-05 2015-11-05 一种伴侣样蛋白的融合蛋白表达载体

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
CN112430273A (zh) * 2019-08-26 2021-03-02 浙江海隆生物科技有限公司 一种狂犬病毒表面的亚单位融合蛋白mG及其制备方法和应用

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CN105924532A (zh) * 2016-06-08 2016-09-07 盘古基因生物工程(南京)股份有限公司 一种可溶性碱性成纤维细胞生长因子融合蛋白表达载体及其应用
CN106084066A (zh) * 2016-06-08 2016-11-09 盘古基因生物工程(南京)股份有限公司 一种可溶性表皮生长因子融合蛋白表达载体及其应用
CN110511951B (zh) * 2019-07-29 2022-03-08 因之彩生物科技（武汉）有限公司 Plb蛋白在构建具有类伴侣样蛋白作用的融合蛋白表达载体中的应用

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WO1999027363A1 (fr) * 1997-11-26 1999-06-03 Tanabe Seiyaku Co., Ltd. Procede d'examen de maladies du rein
WO2008077406A2 (fr) * 2006-12-22 2008-07-03 Aalborg Universitet Couplage d'éléments

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112430273A (zh) * 2019-08-26 2021-03-02 浙江海隆生物科技有限公司 一种狂犬病毒表面的亚单位融合蛋白mG及其制备方法和应用

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