Cloning expression and sequence analysis of heat shock protein 20 of Musca domestica

ObjectiveTo analyze the structural characteristics of heat shock protein 20(HSP20) from Musca domestica and to clone and express the novel gene HSP20.MethodsUsing bioinformatics software package and tools of bioinformatics at web sites of National Center for Biotechnology Information(NCBI),ExPaSy,and combining other bioinformatics software packages,we identified full-length genes encoding HSP20 proteins from the Musca domestica genome library of GenBank,then predicted and analyzed the characteristics of the deduced proteins.The genes encoding HSP20 was amplified with PCR,and cloned into a prokaryotic expression vector PEASY-E1.The recombinant plasmids were transformed into E.coli OrigmiB/DE3 and then the expression of the proteins was induced by isopropyl-beta-D-thiogalactopyranoside(IPTG).The recombinant proteins were purified by Ni-IDA affinity chromatography and tested with sodium dodecyl sulfate polyacrylamide gel electophoresis(SDS-PAGE).ResultsThe full length sequence was 865 bp,containing an open reading frame(ORF)of 567 bp,encoding 188 amino acids with a predicted molecular weight of 21443.2 Da and an isoelectric point of 5.96.PCR,double enzyme digestion and DNA sequencing confirmed that the recombinant expression plasmid was successfully constructed.With SDS-PAGE,the recombinant plasmid PEASY-E1-SP20 was expressed and purified in OrigmiB/DE3,and the molecular weight of 21443.2 Da was noted in HSP20 protein bands.The highest amount of protein expression was generated 12 hours after the induction.The size of protein obtained by SDS-PAGE corresponded with target protein.ConclusionA novel gene coding HSP20 of Musca domestica was cloned,expressed,purified successfully.The purified protein of HSP20 will be of importance for further research in biological function of the gene.