Hostomsky Z, Appelt K, Ogden R C
Agouron Pharmaceuticals Inc., La Jolla, CA 92037.
Biochem Biophys Res Commun. 1989 Jun 30;161(3):1056-63. doi: 10.1016/0006-291x(89)91350-8.
A synthetic gene coding for HIV-1 protease (PR) has been constructed and a system for its efficient expression in E. coli has been established: PR is synthesized as a fusion protein with E. coli dihydrofolate reductase under the control of a bacteriophage T7 promoter. The synthetic gene was constructed to enable rapid construction of defined mutants by restriction fragment replacement. A set of mutants has been constructed which may facilitate elucidation of the mechanism of PR self-cleavage from polyprotein precursors. We have demonstrated that the C-terminal residue (Phe99 in the native sequence) of the processing intermediate is absolutely required for subsequent cleavage at the N-terminal cleavage site. The potential structural role of this residue is discussed with reference to the recently published HIV-1 PR structure.
已构建了一个编码HIV-1蛋白酶（PR）的合成基因，并建立了一种在大肠杆菌中高效表达该基因的系统：PR在噬菌体T7启动子的控制下作为与大肠杆菌二氢叶酸还原酶的融合蛋白进行合成。构建合成基因是为了通过限制性片段置换快速构建特定的突变体。已构建了一组突变体，这可能有助于阐明PR从多蛋白前体中自我切割的机制。我们已经证明，加工中间体的C末端残基（天然序列中的Phe99）对于随后在N末端切割位点的切割是绝对必需的。参照最近发表的HIV-1 PR结构讨论了该残基的潜在结构作用。
