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High-level expression, purification, and solid-state NMR structural studies of antibacterial peptide, bovine lactophoricin

2008년 8월 12일 11시 44분 19초
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목 <발표Ⅰ>
저자 및
박태준, 김용애
한국외국어대학교 화학과, Korea
Lactophoricin (LPcin-I) is a 23-amino acid peptide corresponding to the carboxyterminal 113 to 135 region of component-3 of proteose peptone (PP3) which is a minor phosphoglycoprotein found in bovine milk. It has been reported that lactophoricin have an antibacterial activity with a cationic amphipathic helical structure, but its shorter analogous peptide (LPcin-II), 17-amino acid peptide, corresponding to the 119 to 135 region of PP3 does not display an antibacterial activity any more. In fact, it was interestingly known that LPcin-I, II both interact with phospholipids, but only LPcin-I can incorporate into planar lipid bilayers by forming voltage-dependent channels. In this study, to understand the structure-activity relationship of these peptides, we cloned and expressed two recombinant peptides in the form of inclusion bodies as a ketosteroid isomerase (KSI) fusion protein in Escherichia coli. Fusion proteins were purified by Ni-NTA affinity chromatography under denaturing condition, and recombinant LPcin-I, II were released from the fusion by CNBr cleavage. Final purification of LPcin-I,d LPcin-II was achieved by preparative reversed-phase high performance liquid chromatography (prep-HPLC). Using these methods, we can get several tens milligram quantities of uniformly or selectively 15N labeled peptides, which is enough to measure the solid-state NMR spectroscopy. The peptides were identified by tris-tricine polyacrylamide gel electrophoresis and one- and two-dimensional solid-state NMR spectra of LPcin-I, II were acquired.