Cloning of 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase Gene from Vanda Mimi Palmer and Its Heterologous Expression in Escherichia coli

摘要

Plant 3-hydroxy-3-methylglutaryl-CoA-reductase (HMGR) is involved in the conversion of HMG-CoA into mevalonate (MVA), which yields a biologically active isoprenoid precursor, isopentenyl pyrophosphate (IPP) unit. To date, heterologous expression of HMGR isolated from orchidaceae family has not been reported. The aims of this study were to isolate, clone, over-express and functionally characterize the cDNA encoding a 3-hydroxy-3-methylglutaryl-CoA-reductase of Vanda Mimi Palmer ( VMPHMGR ) in Escherichia coli strain BL21 (DE3) pLysS. The deduced VMPHMGR contains an open reading frame (ORF) of 1689 bp and generates a protein of 562 amino acids with a calculated molecular mass of 59780 Da and a predicted pI value of 6.63, which is 76% identical to other plant HMGRs. Expression analysis of VMPHMGR transcript by real-time RT-PCR showed that it was differentially regulated. Primers with appropriate restriction sites were used to amplify and facilitate in-frame cloning of the VMPHMGR into pET32(a). The expression of VMPHMGR , in E. coli , fused to N-terminal thioredoxin (Trx·Tag), S·Tag and His·Tag fusion proteins in pET32(a) yielded a partially soluble recombinant protein. This expressed VMPHMGR was subjected to functional enzymatic assay and GCMS analysis of the end products detected dehydromevalonic lactone and pantolactone, which were derivatives of mevalonate lactone.