Huo Juan, Shi Haifeng, Yao Qin, Chen Huiqin, Wang Lin, Chen Keping
Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu 212013, PR China.
Protein Expr Purif. 2010 Jul;72(1):95-100. doi: 10.1016/j.pep.2010.01.014. Epub 2010 Jan 20.
Dihydrolipoamide dehydrogenase (DLDH), a flavin-dependent oxidoreductase is essential for energy metabolism. As an oxidoreductase it catalyzes the NAD(+)-dependent oxidation of dihydrolipoamide. In this study, a putative Bombyx mori dihydrolipoamide dehydrogenase (BmDLDH) gene was cloned, expressed, purified and characterized for the first time. The BmDLDH gene was amplified from a pool of silkworm cDNAs by PCR and cloned into Escherichia coli expression vector pET-28a(+). The recombinant His-tagged BmDLDH protein was expressed in E. coli BL21 (DE3) and purified by metal chelating affinity chromatography. The amino acid sequence of recombinant protein was confirmed by mass spectroscopic analysis. Furthermore, the oxidoreductase activity in the reverse reaction indicated that the soluble recombinant BmDLDH produced at lower growth temperature was able to catalyze the lipoamide-dependent oxidation of NADH.
二氢硫辛酰胺脱氢酶（DLDH）是一种黄素依赖性氧化还原酶，对能量代谢至关重要。作为一种氧化还原酶，它催化二氢硫辛酰胺的NAD（+）依赖性氧化。在本研究中，首次克隆、表达、纯化并鉴定了家蚕二氢硫辛酰胺脱氢酶（BmDLDH）基因。通过PCR从家蚕cDNA文库中扩增出BmDLDH基因，并将其克隆到大肠杆菌表达载体pET-28a（+）中。重组His标签的BmDLDH蛋白在大肠杆菌BL21（DE3）中表达，并通过金属螯合亲和层析进行纯化。通过质谱分析确认了重组蛋白的氨基酸序列。此外，反向反应中的氧化还原酶活性表明，在较低生长温度下产生的可溶性重组BmDLDH能够催化NADH的硫辛酰胺依赖性氧化。
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