Cloning, Purification, and Functional Characterization of Bacterial Lipocalin (Blc) for Erucic Acid Binding

Abstract

Long-chain monounsaturated fatty acids such as erucic acid possess limited aqueous solubility, restricting their transport and bioavailability in biological systems. It is proposed that bacterial lipocalin (Blc), a small β-barrel outer-membrane lipoprotein, has the ability to bind and transport hydrophobic ligands, including lipid-like molecules. Blc homologs from three bacterial species, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae, were cloned, expressed, and purified to assess the ability of Blc to serve as a scaffold for fatty-acid delivery. Blc genes were cloned into the pET15-TV-LIC expression vector after PCR amplification. Recombinant constructs were grown and sequence-verified in E. coli DH5α and then transformed into E. coli BL21 for protein expression. Blc was produced as an N-terminal His-tagged protein, allowing it to be purified from soluble lysate through nickel affinity chromatography, and SDS-PAGE confirmed the purity of the recombinant protein. Collected results proved all three Blc variants were successfully cloned, expressed in soluble form, and purified, with E. coli Blc exhibiting the highest soluble expression levels. These results establish the biochemical foundation required for future characterization of Blc-fatty-acid interactions and support the potential of Blc as a protein scaffold for hydrophobic fatty acids such as erucic acid.