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Method for cooperatively establishing optimal bacillus subtilis protease deletion expression host for target protein

A technology of Bacillus subtilis and Bacillus subtilis, applied in the field of biological high, can solve the problems of inability to meet individual needs and poor applicability

Pending Publication Date: 2019-07-12
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Technical problem: Each recombinant protein has its own unique optimal protease-deficient mutant expression host, but the existing Bacillus subtilis protease-deleted hosts have poor applicability and cannot meet this individual demand

Method used

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  • Method for cooperatively establishing optimal bacillus subtilis protease deletion expression host for target protein
  • Method for cooperatively establishing optimal bacillus subtilis protease deletion expression host for target protein
  • Method for cooperatively establishing optimal bacillus subtilis protease deletion expression host for target protein

Examples

Experimental program
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Effect test

Embodiment 1 9

[0018] Example 1 Construction of nine strains of subtilisin deletion mutants

[0019] Such as figure 2 As shown in a and 2b, using Bacillus subtilis 168 as the starting strain, use the pheS* reverse selection marker (ApplMicrobiol Biotechnol, 2017, doi:10.1007 / s00253-016-7906-9) to delete nprE, aprE, epr, bpr, mpr in sequence , nprB, vpr and wprA eight extracellular protease genes to construct Bacillus subtilis mutant PD8. Taking the deletion of nprE as an example, using the total DNA of Bacillus subtilis 168 as a template, using the primer pair nprE-LF-F / nprE-LF-R (SEQ ID NO.1 / SEQ ID NO.2), nprE-RF-F / nprE- RF-R (SEQ ID NO.3 / SEQ ID NO.4) and nprE-DR-F / nprE-DR-R (SEQ ID NO.5 / SEQ ID NO.6) respectively amplify the upstream homology arm (upstream homologous arm, UHA), direct repeat sequence (direct repeat sequence, DR) and downstream homologous arm (downstream homologous arm, DHA), while using the primer pair nprE-PC-F / nprE-PC-R to plasmid pTPC (Appl Microbiol Biotechnol, 2017...

Embodiment 2

[0021] Example 2 Determining beneficial and harmful extracellular proteases to the expression of chlorothalonil hydrolysis dechlorinase (Chd)

[0022] The chlorothalonil hydrolysis dechlorinase gene chd (NCBI sequence number: GQ292539) was connected to the vector pP43NMK (ApplEnviron Microbiol, 2005, doi: 10.1128 / aem.71.7.4101-4103, NCBI sequence number DQ264732) to obtain the expression vector pP43Chd. The specific construction process is as follows: using the total DNA of the bacterial strain Pseudomonas sp.CTN-3 (JBacteriol, 2010, doi: 10.1128 / JB.01547-09) as a template, use the primer pair chd-F / chd-R (SEQ IDNO.9 / SEQ ID NO.10) to amplify chd; the amplified product was ligated with pP43NMK digested by HindⅢ and PstI in vitro by homologous recombination, and the ligated product was transformed into Escherichia coli DH5α competent cells. Transformants were screened on LB plates and verified by sequencing to obtain pP43Chd. Transform pP43Chd into the above-mentioned nine mut...

Embodiment 3

[0023] Example 3 Determining beneficial and harmful extracellular proteases for recombinant expression of amylase AmyM

[0024] The amylase gene amyM (NCBI sequence number KM114206) was connected to the vector pP43NMK to obtain the expression vector pP43AmyM. The specific construction process is as follows: using the total DNA of the strain Corallococcus sp.EGB (Appl EnvironMicrobiol, 2018, doi: 10.1128 / AEM.00152-18) as a template, using the primer pair amyM-F / amyM-R (SEQID NO.11 / SEQ ID NO.12) amplified amyM, ligated the amplified product with pP43NMK digested by HindⅢ and PstI in vitro homologous recombination, transformed the ligated product into E. Transformants were screened on plates and verified by sequencing. Transform pP43AmyM into the above-mentioned nine mutant strains of Bacillus subtilis (PD8, PD7, PD8CnprE, PD8CaprE, PD8Cepr, PD8Cbpr, PD8Cmpr, PD8CnprB and PD8Cvpr), and ferment and culture the obtained nine expressing strains respectively. The medium used is Supe...

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Abstract

The invention belongs to the technical field of biotechnology and discloses a method for cooperatively establishing a most proper bacillus subtilis protease deletion expression host for target protein. Each recombinant protein has a unique optimal protein deletion mutantion expression host, and an existing bacillus subtilis extracellular protease deletion expression host is poor in applicability and cannot meet the individuation demand. In this way, the method for cooperatively establishing the most proper extracellular protease deletion expression host for the recombinant protein is provided.According to the method, the influence of each extracellular protease of bacillus subtilis on target protein production can be accurately evaluated, the beneficial and harmful extracellular proteasesare determined, by inactivating harmful proteases and reserving beneficial proteases, the optimal host is obtained, and the yield of the target protein is increased maximally.

Description

technical field [0001] The invention belongs to the field of biological high technology, and discloses a method for constructing an optimal subtilisin deletion expression host tailored for a target protein. Background technique [0002] Bacillus subtilis is an important host for recombinant protein expression. The host has the advantages of good safety, strong protein secretion ability and low cost of fermentation and culture. On the other hand, the host also has some disadvantages, one of which is that the bacteria secrete eight extracellular proteases, including two neutral proteases (NprE and NprB), three serine proteases (Epr, Bpr and Vpr), alkaline protease AprE, metalloproteinase Mpr and cell wall protease WprA, these proteases degrade secreted extracellular recombinant proteins and reduce their production. A straightforward solution to this problem is to knock out the genes encoding these proteases. Currently reported protease deletion mutant strains include 1A751 ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75C12R1/125
CPCC12N9/54C12N15/75
Inventor 闫新赵雷真刘圆鑫
Owner NANJING AGRICULTURAL UNIVERSITY
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