Stahlhut M, Li Y, Condra J H, Fu J, Gotlib L, Graham D J, Olsen D B
Department of Biological Chemistry, Merck Research Laboratories, Division of Merck & Co., Inc., West Point, Pennsylvania 19486.
Protein Expr Purif. 1994 Dec;5(6):614-21. doi: 10.1006/prep.1994.1084.
Wild-type and several mutant forms of recombinant human immunodeficiency virus type-1 reverse transcriptase were overexpressed as either the p66 or the p51 subunit in a protease-deficient strain of Escherichia coli. Immediately prior to cell lysis, p51 cell paste was mixed with cell paste containing the corresponding overexpressed p66 subunit in a ratio resulting in an excess of the smaller subunit with respect to the larger. During the subsequent chromatography steps stable heterodimer p66/p51 was purified to homogeneity. This protein was characterized by amino acid analysis, denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, analytical gel filtration HPLC, laser desorption mass spectroscopy, and isoelectric focusing. In addition, we were able to obtain crystals of the purified enzyme complexed with a quinazolinone class nonnucleoside inhibitor that diffracted to 3.2 A resolution. A potential application of this expression/purification methodology is the ability to alter specific amino acids residues, by site-directed-mutagenesis, of only one subunit of the RT-dimer.
野生型及几种重组人免疫缺陷病毒1型逆转录酶的突变形式，在蛋白酶缺陷型大肠杆菌菌株中作为p66或p51亚基进行过表达。就在细胞裂解之前，将p51细胞糊与含有相应过表达p66亚基的细胞糊按一定比例混合，使得较小亚基相对于较大亚基过量。在随后的层析步骤中，稳定的异二聚体p66/p51被纯化至同质。该蛋白质通过氨基酸分析、变性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、分析凝胶过滤HPLC、激光解吸质谱和等电聚焦进行表征。此外，我们能够获得与喹唑啉酮类非核苷抑制剂复合的纯化酶的晶体，其衍射分辨率达到3.2 Å。这种表达/纯化方法的一个潜在应用是能够通过定点诱变改变RT二聚体中仅一个亚基的特定氨基酸残基。
