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An expression cassette for efficiently secreting and expressing human fgf21 protein and its application

A technology for expressing cassettes and proteins, which is applied in the biological field and can solve the problems of large consumption, unfavorable energy saving and emission reduction, slow growth, etc.

Active Publication Date: 2021-12-10
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the expression of FGF21 is mainly carried out in Escherichia coli and yeast. As a host cell for protein expression, Escherichia coli has low secretion efficiency, endotoxin, and has caused immune reactions and other unfavorable factors, which will increase the cost of subsequent protein purification processes; Yeast Bacteria, as a fungus, has good protein secretion ability, but the optimum growth temperature is generally around 30°C, the growth is slow and a large amount of cooling water is required in the fermentation process, which is not conducive to energy saving and emission reduction
In addition, the currently commonly used yeast expression system generally uses methanol as an inducer and carbon source, which has certain hidden dangers in production safety, thus further increasing production costs.

Method used

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  • An expression cassette for efficiently secreting and expressing human fgf21 protein and its application
  • An expression cassette for efficiently secreting and expressing human fgf21 protein and its application
  • An expression cassette for efficiently secreting and expressing human fgf21 protein and its application

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preparation example Construction

[0038] 3. Preparation and transformation of DNA:

[0039] DNA was isolated from E. coli and B. subtilis or from agarose gels using DNA preparation kits from Tiangen or Omega according to the manufacturer's instructions. Standard molecular techniques were used in all examples. E. coli was transformed using plasmid DNA as described by Chung C.T. et al., Proc. Natl. Acad. Sci. USA 86, 1989, 21722175. According to the modified "Paris method" (Harwood C.R. Molecular Biological Methods for Bacillus, 1990, John Wiley & Sons Ltd., England), plasmid DNA or DNA fragments were used to transform Bacillus subtilis.

[0040] 4. Bacillus subtilis gene traceless knockout:

[0041] Gene knockout was performed according to the improved "AraR-based Bacillus subtilis gene knockout method" (Liu S, Endo K, et al. Microbiology. 2008; 154(Pt 9): 2562-2570.). First, using the principle of homologous recombination, the spectinomycin resistance gene fragment "Para-spc" carrying the homology arm and r...

Embodiment 1

[0056] Example 1. Construction of rhFGF21 expression cassette containing cistron cistron Optimizing rhFGF21 translation efficiency to promote protein expression level

[0057] 1.1 Design of cistron-containing rhFGF21 expression cassette

[0058] In order to increase the expression level of rhFGF21, the present invention inserts the cistron cistron into the rhFGF21 expression vector to form a rhFGF21 expression cassette containing the cistron cistron. The purpose of inserting cistron is to change the secondary structure of mRNA. RBS is exposed from the original stem-loop structure, which is easy to recruit more ribosomes, enhances the combination of ribosomes and RBS, and improves the translation efficiency of proteins. figure 1 It is a schematic diagram of the structure of the rhFGF21 expression cassette containing the cistron. figure 1 The structure of A is: promoter-RBS-cistron(1-5)-RBS-rhFGF21-terminator, cistron1-cistron5 are the first 18bp of gfp, luc, glvA, rdpe, gsiB g...

Embodiment 2

[0062] Example 2. Helping rhFGF21 to fold correctly by optimizing disulfide bonds to obtain high-yield soluble expression

[0063] The rhFGF21 protein contains a disulfide bond inside. Since the types and expression levels of disulfide bond-related foldases in Bacillus subtilis are relatively low, there are risks such as incorrect formation or formation of misfolded disulfide bonds in the FGF21 protein. Aiming at this situation, the present invention uses the pDL vector (derived from BGSC) as the backbone to clone and construct the integrated vector pDF-d carrying the oxidoreductase DsbA (the construction method is the same as in Example 1) and construct the weakened thioredoxin The carrier ptrx1 of the protein TrxA, that is, the promoter of the gene TrxA is replaced by the promoter P with weaker expression strength spac (construction method is the same as embodiment 1). The vectors pDF-d and ptrx1 were respectively transformed into Bacillus subtilis 1A751, and the mutant str...

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Abstract

The invention belongs to the technical field of molecular biology, and in particular relates to a method for highly expressing recombinant human fibroblast growth factor 21 (rhFGF21) gene by using bacillus subtilis. First, the present invention adjusts the transcription and translation efficiency of the target protein gene by adding different types of cistron cistron sequences at the 5' end of the target protein gene, thereby optimizing its soluble expression level, which can be applied to optimize the expression of different types of heterologous proteins . Secondly, the present invention also optimizes the disulfide bond-related genes to construct the rhFGF21 disulfide bond folding efficiency; overexpresses the molecular chaperone system to construct the protein folding efficiency suitable for rhFGF21; The stable Bacillus subtilis genetically engineered strain finally provides a kind of Bacillus subtilis genetically engineered strain capable of efficiently secreting and expressing rhFGF21 protein with good application prospects.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for highly expressing recombinant human fibroblast growth factor 21 (rhFGF21) protein using bacillus. Background technique [0002] Protein expression technology is one of the core technologies of modern biology. Protein expression can not only be used in biological research, but also provide commercial protein products, such as recombinant vaccines, recombinant insulin, cytokines and other products. Currently commonly used expression systems include Escherichia coli, yeast, insect cells and mammalian cells, etc., but each of them has obvious advantages and disadvantages. Escherichia coli expression system is the most well-studied and has a variety of options. The most commonly used is Novagen's pET expression system, which uses phage T7 RNA polymerase to specifically transcribe the target gene behind the T7 promoter. Although it has the advantages of high expression effici...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/75C12P21/02C12N1/21C12R1/07
CPCC07K14/50C12P21/02
Inventor 张大伟付刚李丹丹
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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