Cloning And Expression Of Lovastatin Synthesis Regulatory Genes LovE And MkH

ObjectiveTo clone Aspergillus terrus and Monascus lovastatin biosynthesis regulatory genes lovE and mkH,analyze and align the sequences; To express and purify lovastatin biosynthesis transcription factor lovE; To construct two eukaryotic recombinant expression vectors of lovE gene.MethodsAccording to well-known lovastatin synthase gene sequences from genebank, primers were designed to amplify from genomic DNA and clone lovastatin biosynthesis regulatory genes lovE and mkH into vector pMD19T-simple. Alignment and analysis of sequencing results lovE,mkH and their encoding amino acids sequences were performed through internet resources and some softwares like DNAMAN. According to well-known Aspergillus terrus lovastatin synthase gene sequences from genebank, primers were designed to amplify from genomic DNA and clone lovE into prokaryotic expression vector pET-21b. LovE-His fusion protein was expressed under IPTG induction and purified by Ni-NTA His tag agarose column. According to well-known Aspergillus terrus lovastatin synthase gene sequences from genebank, primers were designed to amplify from genomic DNA and clone lovE into eukaryotic expression vector pBC-hygro. ResultsTarget fragments lovE and mkH, 1512bp and 1464bp in length respectively, were amplified successfully; Prokaryotic recombinant expression vector pET-lovE was constructed successfully, then fusion protein lovE-His, almost 58KDa in molecular weight, was expressed and purified to electrophoresis purity; Eukaryotic recombinant expression vectors pBC-lovE and pBC-lovElong were constructed successfully.ConclusionHighly homologous lovE and mkH, almost the same as related well-known sequences in genebank, are regulatory genes and their encoding proteins are GAL4-like transcriptional factors. Construction of prokaryotic recombinant expression vector pET–lovE, expression and purification of the fusion protein with high efficiency provide the experiment basis for further understanding of biological function and mechanism of this kind of transcription factor. Construction of eukaryotic recombinant expression vectors pBC–lovE and pBC–lovElong provides the experiment basis for eukaryotic transformation system of the lovE gene.