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Cloning and purification of recombinant silkworm dihydrolipoamide dehydrogenase expressed in Escherichia coli

Juan Huo, Haifeng Shi, Qin Yao, Huiqin Chen, Lin Wang, and Keping Chen

Protein Expr Purif 72(1):6 (2010) PMID 20093189

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.

Copyright © 2010 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.pep.2010.01.014
Version: za2963e q8zaa q8zb6 q8zc3 q8zde q8ze0 q8zf0 q8zg7

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