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Method for promoting extracellular expression of protein in bacillus subtilis by utilizing cutinase

A technology of Bacillus subtilis and cutinase, applied in the field of extracellular expression, can solve problems such as insecurity, and achieve the effects of cost saving, simplified purification process, and efficient extracellular preparation

Active Publication Date: 2021-02-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Aiming at the unsafe problem existing in the extracellular expression of natural intracellular localized protein by using E. coli expression system in the prior art, the present invention aims to obtain a safe and efficient method for extracellular secretion of natural intracellular localized protein , firstly a cutinase mutant is provided, the cutinase mutant is the amino acid sequence shown in SEQ ID NO. Mutations at one or more sites

Method used

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  • Method for promoting extracellular expression of protein in bacillus subtilis by utilizing cutinase
  • Method for promoting extracellular expression of protein in bacillus subtilis by utilizing cutinase
  • Method for promoting extracellular expression of protein in bacillus subtilis by utilizing cutinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Embodiment 1: Construction of recombinant plasmid

[0075] (1) The plasmid pHYPMLd4P preserved in the laboratory (containing pullulanase pμL and chaperone prsA gene, the construction method is recorded in Zhang Kang’s doctoral dissertation of Jiangnan University "Bacillus subtilis strain transformation, promoter optimization and pullulanase High-efficiency preparation research", 2018) as a template, respectively design forward and reverse primers:

[0076] pHY300PLK-F1: 5'-AAGCTTGGTAATAAAAAAAACACCTCC-3';

[0077] pHY300PLK-R1: 5'-TCTTGACACTCCTTATTTGATTTTT-3';

[0078] Amplify the expression vector pHY300PLK-prsA fragment;

[0079] (2) Using the plasmid xylA / pET24a(+) preserved in the laboratory (recorded in Chinese invention patent ZL201210581801.2) as a template, design forward and reverse primers respectively:

[0080] xylA-F: 5'-GGAGTGTCAAGAATGAGCAACTACCAGCCCACAC-3';

[0081] xylA-R: 5'-TTTATTACCAAGCTTTTAGCGCACGCCCAGGAGGTAG-3';

[0082] Amplify the xylose isomer...

Embodiment 2

[0094] Embodiment 2: Construction of cutinase mutant

[0095] Using the recombinant plasmid pHY300PLK-xylA-cut obtained in step (4) in Example 1 as a template, according to the cutinase gene sequence, respectively design and synthesize L175A / T177A, T207A / F209A, I213A / P214A, I178A, L175A, The primers for T177A, T207A, F209A, I213A and P214A mutations were used for site-directed mutation of the cutinase gene and verified by sequencing to obtain the recombinant expression vectors pHY300PLK-xylA-L175A / T177A, pHY300PLK-xylA-T207A / F209A,pHY300PLK-xylA-I213A / P214A,pHY300PLK-xylA-I178A,pHY300PLK-xylA-L175A,pHY300PLK-xylA-T177A,pHY300PLK-xylA-T207A,pHY300PLK-xylA-F209A,pHY300PLK-xylA-I213A,pHY300PLK-xylA- P214A.

[0096] The site-directed mutagenesis primers for introducing the L175A / T177A mutation are:

[0097] L175A / T177A-F:5'-GATCATCGGGGCCGAC GCA GAC GCG ATCGCGCCGGTCG-3'

[0098] L175A / T177A-R:5'-CGACCGGCGCGAT CGC GTC TGC GTCGGCCCCGATGATC-3'

[0099] The site-directed mu...

Embodiment 3

[0126] Example 3: Construction of recombinant bacteria co-expressed with cutinase mutant and xylose isomerase

[0127] (1) Preparation of competent cells:

[0128] Dip the frozen Bacillus subtilis WS5 with an inoculation loop, then streak on the LB plate, and culture overnight at 37°C for activation; pick a single colony and inoculate it in 10mL LB liquid medium, culture it overnight at 37°C, 200rpm for 8h; take 2.5mL Transfer to 40mL LB medium containing 0.5M sorbitol, culture at 37°C with shaking at 200rpm for 4-5h; bathe the bacteria in ice water for 10min, then centrifuge at 5000rpm at 4°C for 5min to collect the bacteria. Resuspend the cells with 50 mL of pre-cooled electroporation buffer, centrifuge at 5000 rpm at 4°C for 5 minutes, remove the supernatant, and rinse 4 times in this way; resuspend the washed cells in 1 mL electroporation medium, and distribute them into 1.5 mL EP tubes , 200 μL per tube to obtain competent cells.

[0129] (2) Transformation of competent...

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Abstract

The invention discloses a method for promoting extracellular expression of protein in bacillus subtilis by utilizing cutinase, and belongs to the technical field of bioengineering, enzyme engineeringand microbial engineering. According to the invention, a cutinase mutant and the target protein are co-expressed in the bacillus subtilis; the target protein comprises xylose isomerase, 4,6-alpha-glucosyltransferase, 4-alpha-glucosyltransferase, trehalose synthase and branching enzyme; extracellular expression of the intracellularly localized target protein can be realized, so that the productionefficiency is improved and the post-extraction process is simplified; and the method has relatively high academic significance and application value.

Description

technical field [0001] The invention relates to a method for promoting extracellular expression of proteins in Bacillus subtilis by using cutinase, and belongs to the technical fields of genetic engineering, enzyme engineering and microbial engineering. Background technique [0002] The extracellular expression of protein can simplify the downstream purification process, save costs, and has absolute advantages in large-scale industrial production. For natural intracellular localized proteins, usually they can only be expressed in cells, and the cells need to be broken up by physical or chemical methods to obtain the target protein. The subsequent extraction process is not only cumbersome but also costly. Downstream extraction steps reduce the cost of product purification has become the goal of researchers. [0003] In the previous research, the inventor's research group found that cutinase achieved high-efficiency "secretion" expression of the extracellular matrix in the E....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55C12N1/21C12N15/75C12R1/125
CPCC12N9/18C12Y301/01074C12Y503/01005C12Y504/99016C12N9/1048C12N9/1051C12N9/90C12N9/92C12N15/75C12Y204/01245C12Y204/01161
Inventor 吴敬宿玲恰黄燕
Owner JIANGNAN UNIV
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