Channelpedia

PubMed 25448388


Referenced in Channelpedia wiki pages of: none

Automatically associated channels: Kv1.3 , Kv1.5



Title: Soluble prokaryotic expression and purification of crotamine using an N-terminal maltose-binding protein tag.

Authors: Thu Trang Thi Vu, Boram Jeong, Jing Yu, Bon-Kyung Koo, Su-Hyun Jo, Robert Charles Robinson, Han Choe

Journal, date & volume: Toxicon, 2014 Dec 15 , 92, 157-65

PubMed link: http://www.ncbi.nlm.nih.gov/pubmed/25448388


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.