title: Soluble prokaryotic expression and purification of crotamine using an N-terminal maltose-binding protein tag.
abstract: Crotamine is a peptide toxin found in the venom of the rattlesnake Crotalus durissus terrificus. Interestingly, crotamine demonstrates promising anticancer, antimicrobial, and antifungal activities. The crotamine peptide can also deliver plasmids into rapidly dividing cells, such as cancer and stem cells, and demonstrates potent analgesic effects. Efficiently producing crotamine in mammalian cells is difficult because it is both cell-permeable and cytotoxic. Prokaryotic expression of this peptide is also difficult to maintain because it does not fold properly in the cytoplasm, resulting in aggregation and in the formation of inclusion bodies. In our current study, we show for the first time that N-terminal fusion with three protein tags-N-utilization substance protein A (NusA), protein disulfide isomerase b'a' domain (PDIb'a'), and maltose-binding protein (MBP)-enables the soluble overexpression of crotamine in the cytoplasm of Escherichia coli. MBP-tagged crotamine was purified using Ni affinity, anion exchange, and MBP chromatography. The tag was cleaved using TEV protease, and the final product was pure on a silver-stained gels. In total, 0.9 mg pure crotamine was obtained from each liter of bacterial culture with endotoxin level approximately 0.15 EU/μg, which is low enough to use in biomedical applications. The identity and intramolecular disulfide bonds were confirmed using MALDI-TOF MS analysis. Purified crotamine inhibited the hKv1.3 channel (but not hKv1.5) in a dose-dependent manner with IC50 value of 67.2 ± 44.7 nM (n = 10), indicating the correct protein folding. The crotamine product fused with MBP at its N-terminus also inhibited the hKv1.3 channel, suggesting that the N-terminus is not involved in the channel binding of the toxin.
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul 138-736, Republic of Korea.
authors.forename: Thu Trang Thi
authors.initials: TT
authors.lastname: Vu
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul 138-736, Republic of Korea.
authors.forename: Boram
authors.initials: B
authors.lastname: Jeong
authors.affiliation: Department of Physiology, Institute of Bioscience and Biotechnology, Kangwon National University School of Medicine, Chuncheon 200-701, Republic of Korea.
authors.forename: Jing
authors.initials: J
authors.lastname: Yu
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul 138-736, Republic of Korea.
authors.forename: Bon-Kyung
authors.initials: BK
authors.lastname: Koo
authors.affiliation: Department of Physiology, Institute of Bioscience and Biotechnology, Kangwon National University School of Medicine, Chuncheon 200-701, Republic of Korea. Electronic address: suhyunjo@kangwon.ac.kr.
authors.forename: Su-Hyun
authors.initials: SH
authors.lastname: Jo
authors.affiliation: Institute of Molecular and Cell Biology, A*STAR (Agency of Science, Technology and Research), Biopolis, Singapore 138673, Singapore; Department of Biochemistry, National University of Singapore, 8 Medical Drive, Singapore 117597, Singapore. Electronic address: rrobinson@imcb.a-star.edu.sg.
authors.forename: Robert Charles
authors.initials: RC
authors.lastname: Robinson
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul 138-736, Republic of Korea. Electronic address: hchoe@ulsan.ac.kr.
authors.forename: Han
authors.initials: H
authors.lastname: Choe
mesh_terms: D003435:Crotalid Venoms / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli; D020128:Inhibitory Concentration 50; D051664:Kv1.3 Potassium Channel / Q000037:antagonists & inhibitors*; D058619:Maltose-Binding Proteins / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D003435:Crotalid Venoms; D051664:Kv1.3 Potassium Channel; D058619:Maltose-Binding Proteins; C010563:crotamine
keywords: Crotamine; Disulfide bond; Escherichia coli; MALDI-TOF MS; Purification; Soluble overexpression; Voltage-gated potassium (K(v)) channel