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| Study on High-level Expression and Characterization of a V125T V8 Protease Mutant with Tolerance to SDS |
| CHENG Ke-li1, LIU Xiao2, LI Su-xia1 |
1 East China University of Science and Technology, State Key Laboratory of Bioreactor Engineering, Shanghai 200237, China;
2 Shanghai Yaxin Biotechnology Co., Ltd, Shanghai 201108, China |
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Abstract Glutamyl endopeptidase enzyme can cleave specifically the peptide bonds on the carboxyl-terminal side of aspartate and glutamate residues. The gene of V8 (V125T) protease mutant was cloned into plasmid pGEX-4T-3 and then the recombinant plasmid was transformed into E. coli BL21 (DE3). After the fermentation in 50 L fermenter, 50 g/L wet cell was obtained, and the fusion protein was expressed as soluble one, the ratio of expressed aim protein reached to 33%. The fusion protein was purified with GST affinity column, activated by enterokinase, purified with anion-exchange chromatography DEAE-FF, 0.998mg purified aim protein per gram wet cell was obtained, the specific activity was 13.47 U/mg pro. with Z-Phe-Leu-Glu-pNA as a substrate. The total activity recovery rate was 97.9%. The values of Km and Vmax of the recombinant V8(V125T) mutant were 0.339 mmol/L and 16.642 μmol/min respectively. The optimum pH was pH8.0 and was stable from pH4.0 to pH 10.0. The optimum temperature was 45℃, and the protease was stable from 4℃ to 35℃ after incubated for 12 h. At 25℃, the protease activity was affected by some 1 mmol/L metal ions, especially by Fe3+ metal ion. The enzyme activity was not affected by 2 mol/L urea and 1 mmol/L EDTA. More than 90% of total activity was kept when it was in 0.1% SDS for 12 h, in 0.5% SDS for 4 h or in 1% SDS for 1 h. The residual activity still was 80% in 0.5% SDS for 12 h and 64% in 1% SDS for 12 h. The tolerance of recombinant V8(V125T) mutant to SDS was vastly improved compared with the wild-type recombinant V8 protease.
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Received: 17 November 2016
Published: 25 April 2017
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