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

哺乳动物乳汁的特征在于复杂的宿主防御系统，其防止可能遍布婴儿的微生物病原体的生长。然而，人们对抗菌成分的命运以及它们如何相互作用以保护接受者知之甚少。α-乳白蛋白（α-LA）是所有物种的乳中的主要蛋白质之一，并且具有将半乳糖基转移酶的作用改变为乳糖合酶的不寻常性质。另一方面，溶菌酶（LZ）是α-LA的同源蛋白，是多种组织和分泌物中普遍存在的抗菌剂。这两种蛋白质独特地共存于哺乳动物的乳汁中，但它们在乳汁中不同存在的意义是完全未知的。本研究对LZ和α-LA进行了研究，以阐明其相互作用和抗菌协同作用的性质。LZ与α-LA形成的复合物对革兰氏阳性菌和革兰氏阴性菌均有明显的抗菌活性。在生理pH条件下，α-LA结合 ...更多信息 LZ-α LA复合物主要以LZ-α LA异二聚体与LZ同二聚体的形式存在。结果首次证明α-LA与LZ结合形成了一种独特的复合物，具有显著增强的杀菌活性。提示LZ和α-LA在新生儿胃肠道微生物感染的宿主防御系统中起重要作用。此外，本研究还开展了平行遗传学研究，目的是从人乳腺上皮细胞中克隆人LZ（hLZ）和人LA（hLA）的cDNA，建立两种人蛋白的天然信号肽在毕赤酵母中的高效表达系统，并设计稳定的hLZ和hLA的框内融合构建体。在甲醇毕赤酵母中成功表达了人LZ、α-LA及其LZ-α-LA融合蛋白，表达量超过20 mg/L，为设计潜在的抗感染药物提供了机会。少

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