Cloning, expression and purification of human S-adenosylmethionine decarboxylase gene alpha subunit.
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 alpha subunit. Clone vector pMD18-T-SAMDC-alpha was successfully constructed by using T-A clone technique. pMD18-T-SAMDC-alpha and pTriEx-4 were digested by NcoI and XhoI double enzymes. The purified SAMDC-alpha fragment was subcloned into the expression vector pTriEx-4 to construct the prokaryotic expression plasmid pTriEx-4-SAMDC-alpha. The recombinant plasmid pTriEx-4-SAMDC-alpha 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 x His.Tag was purified by Ni-NTA chromatographic column. Then, the purified protein can be applied for further research of the immunity of SAMDC.
Gong
Lei
L
Institute of Biochemistry and Molecular Biology, Medical School of Shandong University, Jinan 250012, Shandong, P R China.
Zhang
Bing
B
Zhang
Yan
Y
Hu
Haiyan
H
Zhao
Zhiyi
Z
Liu
Xianxi
X
D012437:Adenosylmethionine Decarboxylase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D029701:Saccharomyces cerevisiae Proteins; D017421:Sequence Analysis; D014157:Transcription Factors
D007527:Isoenzymes; C075111:SWI6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D012437:Adenosylmethionine Decarboxylase