Craig S P, Yuan L, Kuntz D A, McKerrow J H, Wang C C
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2500-4. doi: 10.1073/pnas.88.6.2500.
The bacterial alkaline phosphatase (phoA) promoter and signal peptide have been used previously to control recombinant expression and secretion of eukaryotic proteins in Escherichia coli. Other reports have shown that this expression system can generate relatively modest levels of active hypoxanthine/guanine phosphoribosyltransferase (HPRT; hypoxanthine phosphoribosyltransferase; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), which carries part of the signal peptide but remains in the cytosol of the bacteria. Herein, the phoA promoter without its associated signal peptide is used to regulate expression of the HPRT of Schistosoma mansoni and the ornithine decarboxylase (ODC; L-ornithine carboxy-lyase, EC 4.1.1.17) of Trypanosoma brucei, two enzymes that have been identified as potential targets for antiparasitic chemotherapy. The levels of recombinant expression range from 20% to 60% of the total bacterial protein, and the majority of both recombinant enzymes was soluble. The specific activity for the recombinant trypanosomal ODC was one-third to two-thirds that of the authentic native enzyme and yields were predicted to be 15-30 mg of active enzyme per liter of bacterial culture. The specific activity for the recombinant schistosomal HPRT was equivalent to that for the native enzyme purified from schistosomes and up to 10 mg of enzymatically active HPRT has been purified from a 0.5-liter culture of treated bacteria. These results represent a break-through in recombinant expression of HPRT and ODC.
细菌碱性磷酸酶（phoA）启动子和信号肽此前已被用于控制真核蛋白在大肠杆菌中的重组表达和分泌。其他报告表明，该表达系统可产生相对适度水平的活性次黄嘌呤/鸟嘌呤磷酸核糖转移酶（HPRT；次黄嘌呤磷酸核糖转移酶；IMP：焦磷酸磷酸核糖转移酶，EC 2.4.2.8），该酶携带部分信号肽但仍保留在细菌的胞质溶胶中。在此，不含其相关信号肽的phoA启动子用于调节曼氏血吸虫的HPRT和布氏锥虫的鸟氨酸脱羧酶（ODC；L-鸟氨酸羧基裂解酶，EC 4.1.1.17）的表达，这两种酶已被确定为抗寄生虫化疗的潜在靶点。重组表达水平占细菌总蛋白的20%至60%，并且两种重组酶中的大多数都是可溶的。重组锥虫ODC的比活性是天然酶的三分之一到三分之二，预计每升细菌培养物可产生15 - 30毫克活性酶。重组血吸虫HPRT的比活性与从血吸虫中纯化的天然酶相当，并且已从0.5升处理过的细菌培养物中纯化出高达10毫克的酶活性HPRT。这些结果代表了HPRT和ODC重组表达方面的一项突破。
