title: Cloning and purification of recombinant silkworm dihydrolipoamide dehydrogenase expressed in Escherichia coli.
abstract: 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.
authors.affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu 212013, PR China.
authors.forename: Juan
authors.initials: J
authors.lastname: Huo
authors.forename: Haifeng
authors.initials: H
authors.lastname: Shi
authors.forename: Qin
authors.initials: Q
authors.lastname: Yao
authors.forename: Huiqin
authors.initials: H
authors.lastname: Chen
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
authors.forename: Keping
authors.initials: K
authors.lastname: Chen
mesh_terms: D000818:Animals; D012831:Bombyx / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D008058:Dihydrolipoamide Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D013058:Mass Spectrometry; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D008058:Dihydrolipoamide Dehydrogenase