Fusion Expression of Antimicrobial Peptide GK1 in Escherichia coli

The antimicrobial peptide GK1, a derivative of cecropin A, shows high antimicrobial activities against Gram-positive and Gram-negative bacteria. To avoid the lethal effects on Escherichia coli host cells during its expression, the GK1 gene was combined with a modified human proinsulin (mhPI) gene, inserted into the vector pET28a to construct the recombinant expression plasmid (pET28a-mhPI-GK1) and then transformed into BL21 (DE3). A fusion protein was expressed and resulted in insoluble inclusion bodies counting 20% (W/W) of total cellular proteins. Recombinant GK1 was cleaved from the mhPI-GK1 fusion protein by cyanogen bromide (CNBr) and purified by cation exchange chromatography and reverse-phase high-performance liquid chromatography. The final concentration of recombinant GK1 was 5.7 mg/L E. coli culture, and the purity was above 97%. Its molecular weight measured using electrospray ionization mass spectrometry (ESI-MS) was 2794 D, similar to that of the synthetic GK1. In addition, they have similar antibacterial activity. The results show that mhPI is capable of mediating high-level GK1 gene expression.