High-efficiency Expression and Activity Identification of SrtA Protein in <i>Escherichia coli</i>

Xiujuan ZHANG; Haiwei YU; Dongqi LIU; Jintong ZHAO; Xianghua XIONG

doi:10.13386/j.issn1002-0306.2020070281

Volume 42 Issue 10

May 2021

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Science and Technology of Food Industry > 2021 > 42(10): 83-88. > DOI: 10.13386/j.issn1002-0306.2020070281

ZHANG Xiujuan, YU Haiwei, LIU Dongqi, et al. High-efficiency Expression and Activity Identification of SrtA Protein in Escherichia coli [J]. Science and Technology of Food Industry, 2021, 42(10): 83−88. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2020070281.

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High-efficiency Expression and Activity Identification of SrtA Protein in Escherichia coli

1.
School of Pharmacy, Harbin University of Commerce, Harbin 150076, China
2.
Institute of Biotechnology, Academy of Military Medical Sciences, Academy of Military Sciences, Beijing 100071, China

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Received Date: July 22, 2020
Available Online: March 15, 2021

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Abstract

Optimize codons according to E. coli preference and synthesize highly active mutant srtA geneto construct the Trx protein co-expression vector pTIG-srtA. Then it was transformed into E. coli BL21(DE3) and induced expression at low temperature, and the SrtA protein was purified by Ni²⁺ column affinity chromatography, and connected LH_N-LPETG and G-H_C protein to detect SrtA protein activity. The results of bacterial liquid PCR identification and determination of sequence comparison showed that the pTIG-srtA plasmid was successfully constructed. The results of SDS-PAGE and Western Blot analysis showed that high soluble expression of SrtA protein was achieved in E. coli, Ni²⁺ column affinity chromatography obtains SrtA protein with a purity greater than 95%. The successful connection of LH_N-LPETG and G-H_C protein indicated that the SrtA protein expressed and purified by the prokaryotic system had good transpeptidase activity.
- Sortase A,
- codon optimization,
- enzyme ligation method,
- botulinum neurotoxin A

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