Methanopyrus sp. SNP6源黄素腺嘌呤二核苷酸合成酶序列分析及在大肠杆菌中的异源表达

Sequence analysis of flavin adenine dinucleotide synthetase from Methanopyrus sp. SNP6 and its heterologous expression in Escherichia coli

Methanopyrus sp.SNP6是一株极端嗜热产甲烷菌,本文将其黄素腺嘌呤二核苷酸合成酶基因(fads)进行生物信息学分析并在大肠杆菌中加以表达。生物信息学分析发现,FAD合成酶由150个氨基酸残基组成,理论分子量为17 049.93,等电点为8.95;三级结构为同源二聚体,每个单体含有五股平行的(折叠,比典型的核苷酸结合折叠少一个β折叠。扩增fads基因,构建重组表达质粒p ET28b(+)-fads,在大肠杆菌BL21(DE3)中诱导表达。实验结果显示,该FAD合成酶能在大肠杆菌中高效表达,但部分蛋白以包涵体形式存在。本研究对Methanopyrus sp.SNP6源FAD合成酶进行了蛋白质序列和结构的分析,并首次实现了其在大肠杆菌中的稳定高效表达,为后续该酶的研究和开发提供了基础。

The sequence and structure of flavin adenine dinucleotide（ FAD） synthetase from an extremely thermophilic methanogen Methanopyrus sp. SNP6 was analyzed and the recombinant protein was expressed in E. coli BL21（ DE3）.Bioinformatics analysis showed that the FAD synthetase consisted of 150 amino acid residues,with theoretical molecular weight of 17 049. 93 Da and isoelectric point of 8. 95. The tertiary structure was a homodimer and each monomer contained five parallel β-sheets which were one sheet less than classical mononucleotide-binding fold. The fads gene was amplified and cloned into plasmid p ET28 b（ ＋）. Next,the recombinant plasmid p ET28 b（ ＋）-fads was transformed into E. coli BL21（ DE3）. The results showed that the FAD synthetase could be successfully expressed,but part of the expressed proteins existed in the form of inclusion bodies. In this study,the FAD synthetase from Methanopyrus sp. SNP6 was expressed for the first time,and it would provide a basis foundation for subsequent characterization and application of this enzyme.