Cloning, Expression and Purification of Human S-Adenosylmethionine Decarboxylase Gene α Subunit

Lei Gong；Bing Zhang；Yan Zhang；Hai-Yan Hu；Zhi-Yi Zhao；Xian-Xi Liu

S-adenosylmethionine decarboxylase ； prokaryotic expression

The Chinese Journal of Physiology

50卷1期（2007 / 02 / 28）

29 - 33

英文

S-adenosylmethionine decarboxylase (SAMDC) is an essential enzyme for the synthesis of spermidine and spermine in the biosynthetic pathway of polyamines. The total RNA was extracted from colon cancer tissue and amplified by reverse-transcription PCR with two primers, which span the coding region of SAMDC α subunit. Clone vector pMD18-T-SAMDC-α was successfully constructed by using T-A clone technique. pMD18-T-SAMDC-α and pTriEx-4 were digested by NcoⅠ and XhoⅠ double enzymes. The purified SAMDC-α fragment was subcloned into the expression vector pTriEx-4 to construct the prokaryotic expression plasmid pTriEx-4-SAMDC-α. The recombinant plasmid pTriEx-4-SAMDC-α was transformed into competence E. coli JM109 (DE3). The bacterium was induced by IPTG and its lysates were loaded directly onto SDS-PAGE. An approximately 32 kDa exogenous protein was observed on the SDS-PAGE. The protein was verified by Western blot with anti His•Tag monoclonal antibody. The fusion protein including 6×His•Tag was purified by Ni-NTA chromatographic column. Then, the purified protein can be applied for further research of the immunity of SAMDC.

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