Cloning expression and sequence analysis of Musca domestica lysozyme

Objective To analyze structural characteristics of the protein from Musca domestica lysozyme(MDLZM)by bioinformatics and to clone and express the novel gene of MDLZM for the study of the recombinant protein.Methods Using bioinformatics software package and tools of bioinformatics at web sites of National Center for Biotechnology Information(NCBI), the Expert Protein Analysis System(ExPaSy), and combining other bioinformatics software packages, the full-length gene encoding MDLZM protein from the Musca domestica genome library of GenBank was identified and then the characteristics of the deduced proteins was analyzed.The genes encoding MDLZM were amplified by PCR, and cloned into a prokaryotic expression vector PEASY-E1.The recombinant plasmids were transformed into E.coli OrigmiB/DE3 and followed by the expression of the proteins induced by isopropyl-beta-D-thiogalactopyranoside(IPTG).The recombinant proteins were purified with Ni 2+-charged imino diacetate(Ni-IDA)affinity chromatography, and detected with sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).Results MDLZM gene sequence contains an open reading frame of 435 bp and encodes 145 amino acids with a predicted molecular weight of 15 958.4 Da and a isoelectric point of 8.65.PCR.Double enzyme digestion and DNA sequencing confirmed that the recombinant expression plasmid was successfully constructed.With SDS-PAGE, the recombinant plasmid PEASY-E1-MDLZM was expressed and purified in OrigmiB/DE3, and the molecular weight of 15 958.4 Da was noted in MDLZM protein bands.The highest amount of protein expression was generated 18 hours after the induction.The size of protein obtained by SDS-PAGE method was consistent with that of the target protein.Conclusion A novel gene coding MDLZM was cloned, expresed, and purified successfully.