Cloning, expression, isotope labeling, purification, and characterization of bovine antimicrobial peptide, lactophoricin in Escherichia coli
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.
Section snippets
Vector construction
The forward and reverse primers encoding the two peptides, composed of residues 113–135 of bovine PP3 (LPcin-I) and residues 119–135 of bovine PP3 (LPcin-II), were synthesized by Integrated DNA Technologies (USA). To construct a tandem peptide construction, the 3′ end of the coding strand included a 3-base ATG extension, and the 3′ end of the non-coding strand included a 3-base TAC extension to create suitable overhangs in the duplex for unidirectional end-to-end self ligation.
Complementary
Construction of expression vectors
Annealed DNAs for expression of LPcin-I and LPcin-II were separated by electrophoresis on 2% agarose gels, and successfully purified from gels. These target peptide coding sequences were ligated to a pET31b(+) vector, and the plasmids so obtained were transformed into the initial cloning host to verify insert numbers and DNA sequences. Insert numbers were confirmed by electrophoresis in 1.5% agarose gel after plasmid purification and restriction digestion. As shown in Fig. 2, we obtained
Conclusions
In summary, we describe for the first time a means of expressing at high level and purifying two cationic amphipathic peptides corresponding to the carboxyterminal 113–135 region (LPcin-I) and to the 119–135 region (LPcin-II) of PP3 in E. coli. These peptides were successfully expressed as fusion proteins containing tandem repeats of LPcin-I or LPcin-II. Usually, the expression, purification, and analysis of peptides or small proteins are difficult. In this study, we devised a highly efficient
Acknowledgments
This work was supported by the GRRC program of Gyeonggi province [PRO7023, Development of Anti-Bacterial Peptides using Lactophorin] and a HUFS Research Fund of 2008. In this study, we used the NMR facility at the Korean Basic Science Institute, which is supported by the Bio-MR Research Program of the Korean Ministry of Science and Technology (E28070).
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