Prokaryotic Expression, Purification and Activity Analysis of Recombinant Carboxypeptidase G2

doi:10.13560/j.cnki.biotech.bull.1985.2016.03.029

Abstract

Abstract: This work is to express carboxypeptidase G2(CPG2)in prokaryotic expression system, purify it and detect its activity in vitro. The gene fragment encoding CPG2 was amplified by PCR, and the prokaryotic expressed plasmid pET-30a-CPG2 was constructed by gene recombinant technology, then it was transformed into Escherichia coli BL21(DE3)for the expression with induction. The target protein from grounded cells was purified by metals chelating affinity chromatogram and anion-exchange chromatography. Majority of the fusion protein was expressed in soluble form, and its specificity was confirmed via SDS-PAGE and Western blotting. The preliminary experimental result showed that the recombinant protein degraded MTX, and the specific activity reached 400 U/mg.

Key words: CPG2, prokaryotic expression, purification, MTX

LI Shu-gang, WANG Yong, ZHANG Wei, XIN Yu, DAN Guo-ping, CHAI Xin-juan, GONG Hui-ying, YU Ting-he. Prokaryotic Expression, Purification and Activity Analysis of Recombinant Carboxypeptidase G2[J]. Biotechnology Bulletin, 2016, 32(3): 184-189.

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