Tanhauser S M, Jewell D A, Tu C K, Silverman D N, Laipis P J
Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, JHM Health Center, Gainesville 32610.
Gene. 1992 Aug 1;117(1):113-7. doi: 10.1016/0378-1119(92)90498-e.
Site-directed mutagenesis is widely used to examine structure/function relationships in proteins. We have designed a bacterial expression vector series which is optimized for efficient site-directed mutagenesis and subsequent protein synthesis without intervening subcloning steps. The vectors, derived from the T7 expression vectors of Studier and his collaborators [Studier et al., Methods Enzymol. 185 (1990) 60-89], are small and have a bacteriophage f1 origin of replication for production of single-stranded (ss) DNA. Both single-site mutants [using ssDNA and mutating oligodeoxyribonucleotides (oligos)] and cassette mutants (mutagenesis of a short region by inserting double-stranded oligos into unique restriction sites) are rapidly synthesized and expressed with these vectors. Vector construction and use are detailed with examples showing the expression of the sequences encoding human carbonic anhydrases II and III. Production levels of greater than 60 mg of protein per liter of culture have been obtained.
定点诱变被广泛用于研究蛋白质的结构/功能关系。我们设计了一系列细菌表达载体，这些载体经过优化，可实现高效的定点诱变及后续蛋白质合成，无需中间的亚克隆步骤。这些载体源自Studier及其合作者的T7表达载体[Studier等人，《酶学方法》185 (1990) 60 - 89]，体积小，具有噬菌体f1复制起点，可用于生产单链（ss）DNA。利用这些载体，单一位点突变体[使用ssDNA和诱变寡脱氧核糖核苷酸（oligos）]和盒式突变体（通过将双链oligos插入独特的限制酶切位点对短区域进行诱变）均可快速合成并表达。文中通过展示编码人碳酸酐酶II和III的序列的表达实例，详细介绍了载体的构建和使用方法。每升培养物已获得超过60毫克蛋白质的产量。
