球形芽孢杆菌S-腺苷甲硫氨酸合成酶基因的克隆、表达与活性鉴定

根据S-腺苷甲硫氨酸合成酶的保守性，利用苏云金芽孢杆菌（Bacillus thuringiensis）基因组中S-腺苷甲硫氨酸合成酶序列信息设计引物。以球形芽孢杆菌（Bacillus sphaericus, B.sph）基因组DNA为模版，扩增获得S-腺苷甲硫氨酸合成酶基因，酶解后插入大肠杆菌表达载体pGEX-6P-1，获得重组质粒pGEX-SAM。序列分析表明，来自球形芽孢杆菌的SAM合成酶在基因水平上与大肠杆菌（K02129），枯草芽孢杆菌（Z99129），酿酒酵母（U17246），小鼠（J05571），人（BC018359）的S-腺苷甲硫氨酸合成酶基因，分别有63.9%，75.4%，58.8%，59.1%，55.7%的同源性。由于一级结构存在明显的差异，我们将该重组的质粒转入大肠杆菌DH5α。通过亲和层析纯化出球形芽孢杆菌S-腺苷甲硫氨酸合成酶。用HPLC对S-腺苷甲硫氨酸合成酶的活性进行了分析。

Cloning and Expression the S-adenosylmethionine synthetase from Bacillus sphaericus

Based on the consensus of the S-adenosylmethionine synthetase(SAM) and the sequence of the Bacillus thuringiensis genome, we designed a pair of primer for the S-adenosylmethionine synthetase gene of the Bacillus sphaericus.The fragment of S-adenosylmethionine synthetase gene of the Bacillus sphaericus was amplified by PCR. Sequence analysis was done after the fragment was inserted into the the expression vector pGEX-6P-1 to construct the plasmid pGEX-SAM. The results show that the homologous of the fragment with the SAM synthetase gene from E. coli, Bacillus subtilis, Saccharomyces cerevisiae, rat and Homo sapiens is 63.9%，75.4%，58.8%，59.1%，55.7% respectively. For the difference between the fragment and the reported SAM synthetase gene, we expressed the gene in E.coli DH5α and the purified 43KD proterin was acquired. The activity of catalyzing the formation of SAM was identified by determining the produced SAM through High Performance Liquid Chromatography.