Potential Antimicrobial Strategy Using Genetically Engineered Homo & Hetero-Dimer of alpha-Lactalbumin and Lysozyme

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Mammalian milk is characterized by a complex host defense system that prevents the growth of microbial pathogens that may pervade the infant. However, little is known of the fate of the antimicrobial components and precisely how they interact to protect the recipient. Alpha-lactalbumin (α-LA) is one of the major proteins in milk of all species and has the unusual property of acting to modify the action of galactosyltransferase to a lactose synthase. On the other hand, lysozyme (LZ), a homologous protein to α-LA, is a ubiquitous antimicrobial in a variety of tissues and secretions. These two proteins uniquely co-exist in mammalian milk but the significance of their distinct presence in milk is completely unknown. In this study, LZ and α-LA were investigated to elucidate the nature of their interaction and antibacterial synergy. A complex of LZ and α-LA exhibited greatly enhanced antibacterial activity against the Gram-positive and -negative bacteria. Under physiological pH, α-LA associa … More ted with LZ, whereas the complex existed mainly as LZ-αLA heterodimer with LZ homodimer. The results provided the first demonstration of association of α-LA with LZ leading to the formation of a distinct complex with significantly enhanced bactericidal activity. Our results strongly suggest the important role of LZ and α-LA in the host defense system of the newborn against gastric microbial infections. Furthermore, a parallel genetic study was initiated with the objectives of cloning the cDNAs of human LZ (hLZ) and human LA (hLA) from human mammary epithelial cells, establish high-level expression system in Pichia under the native signal peptides of the two human proteins, and to design a stable in-frame fusion constructs between hLZ and hLA., Human LZ, α-LA, and their LZ-α-LA fusion construct were successfully produced in Pichia methanolica with expression level exceeding 20 mg/L and thus offer a great opportunity for the design of potential antimicrobial drug in the treatment of infectious diseases. Less