Taylor A, Brown D P, Kadam S, Maus M, Kohlbrenner W E, Weigl D, Turon M C, Katz L
Corporate Molecular Biology, Abbott Laboratories, IL 60064.
Appl Microbiol Biotechnol. 1992 May;37(2):205-10. doi: 10.1007/BF00178172.
A 1.3-kb segment of Escherichia coli DNA containing the regulatory gene, araC, and the promoter of the araBAD operon was amplified by the polymerase chain reaction (PCR) and cloned into pUC18, resulting in plasmid pKB130 that produced the alpha fragment of beta-galactosidase upon addition of L-arabinose (L-ara). A synthetic gene for human immunodeficiency virus (HIV)-1 preprotease was placed downstream of the ara-BAD promoter in pKB130 to create a translational fusion inducible by addition of L-ara. The fusion protein correctly autoprocessed in vivo to yield a mature 99-amino-acid HIV-1 protease, which was found predominantly in inclusion bodies. This material could be refolded to an active form, which was purified to homogeneity. A small fraction of the protease was expressed in vivo as a soluble active form, which allowed the monitoring of expression during fermentation by a rapid and simple whole cell assay employing an HIV-1 protease-specific fluorogenic substrate.
一段包含调节基因araC和araBAD操纵子启动子的1.3千碱基大肠杆菌DNA片段通过聚合酶链反应（PCR）扩增，并克隆到pUC18中，得到质粒pKB130，该质粒在添加L-阿拉伯糖（L-ara）时产生β-半乳糖苷酶的α片段。将人类免疫缺陷病毒（HIV）-1前蛋白酶的合成基因置于pKB130中ara-BAD启动子的下游，以创建通过添加L-ara诱导的翻译融合。融合蛋白在体内正确地自动加工，产生成熟的99个氨基酸的HIV-1蛋白酶，其主要存在于包涵体中。该物质可以重折叠成活性形式，并纯化至同质。一小部分蛋白酶在体内以可溶性活性形式表达，这使得通过使用HIV-1蛋白酶特异性荧光底物的快速简单全细胞测定来监测发酵过程中的表达成为可能。
