High–level expression of housefly cecropin A in Escherichia coli using a fusion protein

ObjectiveTo investigate the effect of utilizing a molecular partner on high-level expression of Musca domestica (M. domestica) cecropin in Escherichia coli (E. coli) and to identify the expressed products.MethodsThe genomic sequence of M. domestica cecropin A (MC) and M. domestica ubiquitin (UBI) were searched from Genbank and amplified by reverse transcriptase polymerase chain reaction (RT-PCR). Two expression plasmids, pET32a-MC and pET32a-UBI-MC, were constructed and transferred into E. coli and were then induced by Isopropyl β-D-1-Thiogalactopyranoside (IPTG). The expression of the fusion proteins Trx-MC and Trx-UBI-MC was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fusion protein Trx-MC was verified by Western blot analysis. The bactericidal activity of the purified MC was quantitatively determined using E. coli BL21(DE3).ResultsThe result showed that the fusion proteins were successively expressed in E. coli BL21 cells. A band at the expected position of 24 kDa representing the Trx-MC target protein was positivelystained, and the band at 4 kDa representing the hydrolysis of mature MC protein was also observed at the expected position. The expression levels of Trx-UBI-MC were higher than that of Trx-MC in E. coli. MC exhibited antimicrobial activity.ConclusionsWith high-level expression of housefly cecropin A in E. coli using a fusion protein, MC exhibited antimicrobial activity.