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.