ORBK (LKGCWTKSIPPKPCFK) is a cyclic cationic peptide that has potent antimicrobial properties and trypsin inhibitory activities. To explore a new approach for expressing ORBK in Escherichia coli, a sequence encoding ORBK was cloned into pET28a vector in which maltose-binding protein (MBP) was used as a fusion partner and an N-terminal 6-His as an affinity tag. Protein expression was induced with 0.5mM Isopropyl-thio-galactoside (IPTG) for 4h at 37°C. The recombinant ORBK was then purified by Ni affinity column and further digested with tobacco etch virus (TEV) enzyme. The cleaved ORBK peptide was separated from MBP fusion partner by reverse phase high performance liquid chromatography (RP-HPLC) and oxidized... More
ORBK (LKGCWTKSIPPKPCFK) is a cyclic cationic peptide that has potent antimicrobial properties and trypsin inhibitory activities. To explore a new approach for expressing ORBK in Escherichia coli, a sequence encoding ORBK was cloned into pET28a vector in which maltose-binding protein (MBP) was used as a fusion partner and an N-terminal 6-His as an affinity tag. Protein expression was induced with 0.5mM Isopropyl-thio-galactoside (IPTG) for 4h at 37°C. The recombinant ORBK was then purified by Ni affinity column and further digested with tobacco etch virus (TEV) enzyme. The cleaved ORBK peptide was separated from MBP fusion partner by reverse phase high performance liquid chromatography (RP-HPLC) and oxidized to obtain the cyclic form. Mass spectroscopy and nuclear magnetic resonance (NMR) spectroscopy were performed for ORBK characterization. Herein we have developed an effective and reliable method to express and purify ORBK which sets a solid foundation for future structural and functional studies.
