摘要
将Methanopyrus sp.SNP6进行功能基因组测序,获得S-腺苷甲硫氨酸合成酶基因(sam)序列,并进行序列分析。将sam基因扩增后连接至表达质粒p ET-28b(+),转化E.coli BL21(DE3),获得重组菌后进行诱导表达。生物信息学分析表明,sam基因编码蛋白的理论分子量为44 086.4Da,三级结构为同源四聚体,与其他相关古菌来源的S-腺苷甲硫氨酸合成酶的蛋白序列较保守。实验结果显示,构建的重组表达质粒p ET28b(+)-sam可在E.coli BL21(DE3)宿主菌中高水平表达。重组蛋白的分子量与预期值基本一致,部分为胞内可溶性表达,另一部分以包涵体形式存在。本研究首次实现了Methanopyrus sp.SNP6菌株S-腺苷甲硫氨酸合成酶的异源表达,为后期的蛋白纯化、酶学性质研究和酶促转化法生产S-腺苷甲硫氨酸奠定了理论基础。
The genome of Methanopyrus sp. SNP6 was sequenced and the sam gene was PCR amplified followed by sequence analysis. The sam gene was cloned into the expression vector p ET-28b（ ＋）,then transformed into Escherichia coli BL21（ DE3）. The recombinant protein was induced for expression by IPTG and the expressed protein was detected by using SDS-PAGE. The results showed that sam gene was 1 194 bp in length,coding for 397 amino acids,its theoretical molecular weight was 44 086. 4 Da and it was a homologous tetramer. Its protein sequence was conservative with some related archaea. SDS-PAGE showed that the recombinant protein could be expressed and the molecular weight was consistent with the expectation. The recombinant protein was intracellular. A part of expressed proteins were solvable and the other was inclusion bodies. In this study,the sam gene was successfully cloned and expressed. It would provide a foundation for further purification, structural and functional research of S-adenosylmethionine synthetase from Methanopyrus sp. SNP6.
出处
《工业微生物》
CAS
2017年第4期24-29,共6页
Industrial Microbiology
基金
国家海洋经济创新发展区域示范项目(12PYY001SF08)
浙江省新苗人才计划项目(2015R403095)
关键词
嗜热古菌
S-腺苷甲硫氨酸合成酶
异源表达
序列分析
thermophilic archaea
S-adenosylmethionine synthetase
sequence analysis
prokaryotic expression
