title: Recombinant expression, purification and characterization of antimicrobial peptide ORBK in Escherichia coli.
abstract: 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.
authors.affiliation: High Magnetic Field Laboratory, Hefei Institutes of Physical Science, Chinese Academy of Science, Hefei, Anhui 230031, PR China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.affiliation: High Magnetic Field Laboratory, Hefei Institutes of Physical Science, Chinese Academy of Science, Hefei, Anhui 230031, PR China.
authors.forename: Jiarong
authors.initials: J
authors.lastname: Wang
authors.affiliation: High Magnetic Field Laboratory, Hefei Institutes of Physical Science, Chinese Academy of Science, Hefei, Anhui 230031, PR China.
authors.forename: Jing
authors.initials: J
authors.lastname: Yang
authors.affiliation: High Magnetic Field Laboratory, Hefei Institutes of Physical Science, Chinese Academy of Science, Hefei, Anhui 230031, PR China.
authors.forename: Chanjuan
authors.initials: C
authors.lastname: Wan
authors.affiliation: High Magnetic Field Laboratory, Hefei Institutes of Physical Science, Chinese Academy of Science, Hefei, Anhui 230031, PR China.
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Wang
authors.affiliation: High Magnetic Field Laboratory, Hefei Institutes of Physical Science, Chinese Academy of Science, Hefei, Anhui 230031, PR China; Center of Medical Physics and Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, PR China. Electronic address: hbsun@hmfl.ac.cn.
authors.forename: Hongbin
authors.initials: H
authors.lastname: Sun
mesh_terms: D000890:Anti-Infective Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D058619:Maltose-Binding Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins
keywords: Antimicrobial peptides; Cyclic peptide; Expression; Purification