Darji A, Chakraborty T, Niebuhr K, Tsonis N, Wehland J, Weiss S
Department of Cell Biology and Immunology, GBF, Braunschweig, Germany.
J Biotechnol. 1995 Dec 15;43(3):205-12. doi: 10.1016/0168-1656(95)00138-7.
Listeriolysin, the hemolysin of the pathogenic species Listeria monocytogenes, was expressed in the non-pathogenic species Listeria innocua. Coexpression of the positive regulatory factor prfA in the plasmid vector in conjunction with the structural gene hly increased the expression over 500-fold. Purification from supernatant fluids was achieved by two steps of ion exchange chromatography. The procedure resulted in over 60% yield of a hemolytically active, homogeneous 58 kDa protein which was used to produce monospecific antibodies. As shown by immunoblot the purified listeriolysin was free of p60, a highly immunogenic protein of similar size also produced by Listeria spp., which otherwise would interfere with immunoassays. Listeriolysin retained full activity for more than 6 months at -70 degrees C.
溶血素，即致病性单核细胞增生李斯特菌的溶血素，在非致病性无害李斯特菌中表达。在质粒载体中与结构基因hly共表达阳性调节因子prfA，使表达增加了500多倍。通过两步离子交换色谱法从上清液中实现纯化。该方法得到了产率超过60%的具有溶血活性的、均一的58 kDa蛋白质，该蛋白质用于制备单特异性抗体。免疫印迹显示，纯化的溶血素不含p60，p60是李斯特菌属也产生的大小相似的高免疫原性蛋白质，否则会干扰免疫测定。溶血素在-70℃下6个月以上仍保持全部活性。
