Cloning, expression, isotope labeling, purification, and characterization of bovine antimicrobial peptide, lactophoricin in Escherichia coli.
Lactophoricin (LPcin-I) is a 23-amino acid peptide that corresponds to the carboxyterminal 113-135 region of component-3 of proteose peptone (PP3), a minor phosphoglycoprotein found in bovine milk. It has been reported that lactophoricin has antibacterial activity and a cationic amphipathic helical structure, but its shorter analogous peptide (LPcin-II), a 17-amino acid peptide, corresponding to the 119-135 region of PP3 does not display antibacterial activity. LPcin-I and LPcin-II have similar charge ratios and identical hydrophobic/hydrophilic sectors, according to their helical wheel projection patterns, and both peptides show cationic amphipathic helical folding and interact with membranes. However, it is known that only LPcin-I incorporates into planar lipidic bilayers to form voltage-dependent channels. In this study, the authors cloned and expressed the two recombinant peptides as ketosteroid isomerase (KSI) fusion proteins inclusion bodies in Escherichia coli. These peptides were subjected to NMR structural studies to explore their structure-activity relationships. Fusion proteins were purified by Ni-NTA affinity chromatography under denaturing conditions, and recombinant LPcin-I and LPcin-II were released from fusion by CNBr cleavage. Final purifications of LPcin-I and LPcin-II were achieved by preparative reversed-phase high performance liquid chromatography. Using these methods, we obtained several tens of milligrams of uniformly and selectively (15)N labeled peptides per liter of growth, which was sufficient for solid-state NMR spectroscopy. Peptides were identified by tris-tricine polyacrylamide gel electrophoresis and HSQC spectra. Initial structural data were obtained by solution NMR spectroscopy and compared in membrane-like environments.
Park
Tae-Joon
TJ
Department of Chemistry and Protein Research Center for Bio-Industry, Hankuk University of Foreign Studies, Yong-In 449-791, Republic of Korea.
Kim
Ji-Sun
JS
Choi
Sung-Sub
SS
Kim
Yongae
Y
D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002417:Cattle; D003001:Cloning, Molecular; D005260:Female; D015870:Gene Expression; D007553:Isotope Labeling / Q000379:methods; D008894:Milk Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D023181:Antimicrobial Cationic Peptides; D008894:Milk Proteins; D011993:Recombinant Fusion Proteins