Prokaryotic overexpression system of recombinant Agrius convolvuli lysozyme produced as inclusion body

We have isolated lysozyme from immunized hemolymph of Agrius convolvuli and amplified the lysozyme cDNA. The cDNA sequence encodes a 120 amino acids mature protein. Agrius lysozyme gene amplified with gene specific primers was ligated into the pGEX 4T-1 vector, which contained the glutathione S-transferase (GST) gene for fusion partner. A recombinant lysozyme was expressed in E. coli Rosetta cells for pGEX 4T-1 expression vector and the fusion protein was induced by IPTG. The recombinant protein produced as inclusion body was resolubilized by the solubilization buffer, and then it was dialyzed in refolding buffer. After thrombin cut, recombinant lysozyme was purified by ion exchange chromatography and reverse phase chromatography. The recombinant lysozyme was identified by SDS-PAGE analysis. We observed immunoreactivity against the anti-Agrius lysozyme by western blot analysis of this protein. The recombinant lysozyme had antibacterial activity against Bacillus megaterium and Micrococcus luteus, which was examined by inhibition zone assay.