Recombinant expression, purification and characterization of antimicrobial peptide ORBK in Escherichia coli.
作者： Yan Li , Jiarong Wang , Jing Yang , Chanjuan Wan , Xiaoming Wang
DOI: 10.1016/J.PEP.2013.12.011
关键词: Affinity chromatography 、 Cyclic peptide 、 Chromatography 、 Tobacco etch virus 、 Escherichia coli 、 Trypsin 、 Biochemistry 、 Recombinant DNA 、 Peptide 、 Antimicrobial peptides 、 Chemistry
摘要: ORBK (LKGCWTKSIPPKPCFK) is a cyclic cationic peptide that has potent antimicrobial properties and trypsin inhibitory activities. To explore new approach for expressing in Escherichia coli, sequence encoding was cloned into pET28a vector which maltose-binding protein (MBP) used as fusion partner an N-terminal 6-His affinity tag. Protein expression induced with 0.5mM Isopropyl-thio-galactoside (IPTG) 4h at 37°C. The recombinant then purified by Ni column further digested tobacco etch virus (TEV) enzyme. cleaved separated from MBP reverse phase high performance liquid chromatography (RP-HPLC) oxidized to obtain the form. Mass spectroscopy nuclear magnetic resonance (NMR) were performed characterization. Herein we have developed effective reliable method express purify sets solid foundation future structural functional studies.
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