期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1990
卷号：87
期号：5
页码：1937-1941
DOI：10.1073/pnas.87.5.1937
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：We report an efficient, general approach for the construction of protein-overproducing strains of Escherichia coli. The method, expression-cassette polymerase chain reaction (ECPCR), allows the insertion of virtually any contiguous coding sequence between sequences that direct high-level protein biosynthesis in E. coli. The gene expression cassettes obtained by ECPCR are inserted into a regulated overexpression plasmid, and the resulting construct is used to transform E. coli. By effecting simultaneous 5' and 3' modification of a coding sequence, ECPCR permits the facile generation of mutant proteins having N- and/or C-terminal truncations. The method is a highly efficient way to dissect a multidomain protein into its component domains. The efficiency of the ECPCR approach is demonstrated in this study by construction of permuted overexpression vectors for the first two extracellular domains of the human CD4 protein.
