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A signal peptide mutant for increasing the secretion of heterologous protein, its construction method and application

A technology of heterologous protein and signal peptide, applied in the field of genetic engineering, can solve the problem of difficulty in large-scale production of heterologous protein, and achieve the effects of significant secretion expression, promoting secretion expression, and increasing expression amount.

Active Publication Date: 2021-01-22
TIANJIN UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the expression of heterologous proteins in the host often forms insoluble inclusion bodies, which are easily degraded by proteases, which make it difficult to produce heterologous proteins on a large scale

Method used

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  • A signal peptide mutant for increasing the secretion of heterologous protein, its construction method and application
  • A signal peptide mutant for increasing the secretion of heterologous protein, its construction method and application
  • A signal peptide mutant for increasing the secretion of heterologous protein, its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1: Preparation of signal peptide mutants and their induction of foreign protein expression

[0068] Using the alkaline protease derived from Bacillus clausii as the reporter gene, the signal peptide derived from Bacillus amyloliquefaciens was transformed to obtain a signal peptide mutant, which increased the secretion of alkaline protease in the host of Bacillus subtilis, in which the signal peptide Refer to the attached structural diagram figure 1 .

[0069] (1) Acquisition of the target gene

[0070] Target gene is alkaline protease gene aprE (alkaline protease amino acid sequence is shown in SEQ ID NO:1, and the nucleotide sequence of protease gene aprE is shown in SEQ ID NO:12, GenBank:FJ940727.1, the construction of target gene Reference patent literature: Invention patent with application number 201910332253.1 "Genetically engineered bacteria for efficient heterologous expression of alkaline protease and its construction method", in which the acquisition...

Embodiment 2

[0101] Example 2: Preparation of signal peptide mutants and their induction of foreign protein expression

[0102] Using the alkaline xylanase derived from Bacillus pumilus as the reporter gene, the signal peptide YwjE derived from Bacillus subtilis was transformed to obtain a signal peptide mutant, which increased the secretion of alkaline xylanase in the host of Bacillus subtilis, The schematic diagram of the signal peptide structure refers to the attached figure 1 .

[0103] (1) Acquisition of the target gene alkaline xylanase gene:

[0104] The target gene alkaline xylanase gene (the amino acid sequence of alkaline xylanase is shown in SEQ ID NO: 5, the nucleotide sequence of alkaline xylanase gene is shown in SEQ ID NO: 13, 687bp, GenBank: KU301789.1) was synthesized by Suzhou Jinweizhi Co., Ltd.

[0105] Construction of recombinant alkaline xylanase genetically engineered bacteria:

[0106] The plasmid containing the alkaline xylanase gene was double digested (BamHI-...

Embodiment 3

[0127] Using the cutinase derived from Fusarium solani as the reporter gene, the signal peptide YjiA derived from Bacillus subtilis was transformed to obtain a signal peptide mutant, which can increase the secretion of cutinase in the host of Bacillus subtilis, and the structure diagram of the signal peptide is referred to attached figure 1 .

[0128] (1) Obtaining the target gene:

[0129] The target gene cutinase gene (the amino acid sequence of cutinase is shown in SEQ ID NO: 8, the nucleotide sequence of cutinase gene is shown in SEQ ID NO: 14, GenBank: M29759.1) was synthesized by Suzhou Jinweizhi Co., Ltd.

[0130] Construction of recombinant cutinase genetically engineered bacteria:

[0131] The plasmid containing the cutinase gene was double digested with (BamHI-SphI), and the 2770bp band was recovered by cutting the gel, and connected to the pWB980 expression vector (such as figure 2 shown), transformed into Bacillus subtilis WB600. Reference patent for the expre...

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Abstract

The invention discloses a signal peptide mutant for improving the secretion amount of heterologous protein, a construction method and application thereof, and belongs to the technical field of genetic engineering. The signal peptide mutant in the present invention is site-directed mutation of the charged amino acid in the N region of the signal peptide derived from the Sec pathway of Gram-positive bacteria, so that the charge density of the amino acid in the N region is between 0.2 and 0.8; The H region of the peptide is mutated so that the hydrophobicity of the amino acids in the H region is between 0 and 70; the amino acids in the C region of the signal peptide are site-directed to mutate, so that the last three amino acids in the C region are mutated to alanine-any amino acid-C amino acid. The invention can effectively increase the secretion amount of the heterologous protein in the microbial host, and effectively promote the high-efficiency expression and industrial production of the target protein.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a signal peptide mutant for increasing the secretion of heterologous protein, its construction method and application. Background technique [0002] Recombinant proteins such as industrial enzymes and biopharmaceutical proteins have a very broad application market. A variety of prokaryotic and eukaryotic expression systems have been developed to produce recombinant proteins. Among them, bacteria have the advantages of easy handling and many genetic manipulation tools, and are more often used as expression hosts for recombinant proteins. However, the expression of heterologous proteins in the host often forms insoluble inclusion bodies, which are easily degraded by proteases, which make it difficult to produce heterologous proteins on a large scale. Secreting the recombinant protein into the growth medium of the bacterial host can effectively prevent the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/32C12N15/31C12N15/75C12N1/21C12N9/24C12N9/18C12R1/125
CPCC07K14/32C12Y301/01074C12N9/18C12N15/75C07K2319/02
Inventor 路福平李玉彭冲牛馨任绍东王兴吉刘逸寒王克芬张会图刘文龙刘夫锋张杰佟新伟
Owner TIANJIN UNIV OF SCI & TECH
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