Cloning, expression, isotope labeling, purification, and characterization of bovine antimicrobial peptide, lactophoricin in Escherichia coli

Abstract

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 ¹⁵N 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.

Introduction

Because bacteria are becoming resistant to conventional antibiotics, researchers are searching for new ways to combat microbial infections [1]. Recently, antimicrobial peptides (AMPs)¹ have received much interest due to their potential uses as new generation antibiotics. Indeed the fact that AMPs are active against a wide range of pathogenic microorganisms makes them attractive candidates for possible use as; antibiotics [2], [3], bio-preservatives [4], wound healing agents [5], [6], anticancer agents [7], [8], and for enhancing disease resistance in aquaculture [9]. Several hundreds of AMPs have been identified in diverse life forms ranging from bacteria, fungi, plants, amphibians, to mammals, including humans [10], [11], [12]. These peptides are believed to kill target cells by disrupting the ordered structures of cell membranes via barrel-stave, carpet, or toroidal mechanisms [13]. Their modes of action differ from those of conventional antibiotics and their broad spectrum antimicrobial activities make the AMPs potentially useful for pharmacological applications and commercial exploitation [14], [15], [16]. However, their mechanisms are not fully understood and more research is required on this topic. Furthermore, research is needed to determine how to; produce them more effectively, increase their serum half-lives, reduce their toxicities, and how to lower their production costs.

Proteose-peptone is found in the heat-stable, acid-soluble protein fraction of milk and has important functional properties. Component-3 of proteose peptone (PP3; also called lactophorin (LP)) is a phosphorylated glycoprotein, which partitions to most hydrophobic fractions. It appears to be largely responsible for the physicochemical properties of proteose-peptones and for their important biological roles in bovine milk [17]. Homologous proteins have been characterized in the milks of other species, such as, camel [18], llama [19], ewe, and goat [20], [21]. However, PP3 has not been found in human milk [22]. Structurally, PP3 contains at least two distinct domains, that is, N-terminal and C-terminal domains. Furthermore, whereas the N-terminal domain (from residue 1 to 97) is largely negatively charged and contains several post-transductional sites, the C-terminal domain (from residue 98 to 135) is positively charged and displays an amphipathic character, if an α-helical structure is assumed [22], [23], [24].

Lactophoricin (LPcin-I) is a cationic amphipathic peptide with 23-amino acid residues, and corresponds to the carboxy terminal 113–135 region of PP3. LPcin-I inhibits the growth of both gram-negative and gram-positive bacteria, but has no hemolytic activity at concentrations of <200 μM [22]. In contrast to LPcin-I, it has been reported that LPcin-II, which corresponds to the 119–135 region of PP3, has no antibacterial activity, which is interesting because LPcin-I and LPcin-II have similar charge ratios and identical hydrophobic/hydrophilic sectors based on the helical wheel pattern [24], [25], [26]. Furthermore, both peptides show cationic and interact with phospholipids. Nevertheless, only LPcin-I is incorporated into planar lipidic bilayers, in which it forms voltage-dependent channels [26].

To obtain structural information on LPcin-I and LPcin-II, a convenient method for obtaining milligram quantities of isotopically labeled peptides was developed. In addition, in order to understand the antibacterial mechanism of LPcin-I and structure-activity relationships between LPcin-I and LPcin-II, we cloned and expressed two recombinant peptides in the form of inclusion bodies as a ketosteroid isomerase (KSI) fusion proteins in Escherichia coli. To achieve high yields of the recombinant peptides, we adopt a cloning strategy involving the insertion of tandem repeats of peptide coding regions into an expression vector [27]. Uniformly and selectively ¹⁵N labeled KSI fusion proteins were overexpressed at high levels, and the C-terminal His-tagging allowed fusion proteins to be purified by Ni–NTA immobilized metal affinity chromatography under denaturing conditions. Subsequently, KSI fusion partners and His-tags were easily removed by CNBr cleavage in 70% formic acid. Final purifications of LPcin-I and LPcin-II were achieved by preparative reversed-phase high performance liquid chromatography (prep-HPLC). Using the methods described, we prepared uniformly and selectively ¹⁵N labeled peptides at >10 mg/L levels in M9 minimal media, which was sufficient for solid-state NMR spectroscopy. The two recombinant peptides were characterized by tris-tricine polyacrylamide gel electrophoresis and initial structural data were obtained by solution NMR spectroscopy and compared in membrane-like environments.