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MacFerrin KD, Terranova MP, Schreiber SL, et al. Overproduction and dissection of proteins by the expression-cassette polymerase chain reaction. Proc Natl Acad Sci U S A. 1990;87(5):1937-41doi: 10.1073/pnas.87.5.1937.
MacFerrin, K. D., Terranova, M. P., Schreiber, S. L., & Verdine, G. L. (1990). Overproduction and dissection of proteins by the expression-cassette polymerase chain reaction. Proceedings of the National Academy of Sciences of the United States of America, 87(5), 1937-41. https://doi.org/10.1073/pnas.87.5.1937
MacFerrin, K D, et al. "Overproduction and dissection of proteins by the expression-cassette polymerase chain reaction." Proceedings of the National Academy of Sciences of the United States of America vol. 87,5 (1990): 1937-41. doi: https://doi.org/10.1073/pnas.87.5.1937
MacFerrin KD, Terranova MP, Schreiber SL, Verdine GL. Overproduction and dissection of proteins by the expression-cassette polymerase chain reaction. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1937-41. doi: 10.1073/pnas.87.5.1937. PMID: 2408046; PMCID: PMC53599.
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Proc Natl Acad Sci U S A. 1990 Mar;87(5):1937-41. doi: 10.1073/pnas.87.5.1937.

Overproduction and dissection of proteins by the expression-cassette polymerase chain reaction.

Proceedings of the National Academy of Sciences of the United States of America

K D MacFerrin, M P Terranova, S L Schreiber, G L Verdine

Affiliations

  1. Department of Chemistry, Harvard University, Cambridge, MA 02138.

PMID: 2408046 PMCID: PMC53599 DOI: 10.1073/pnas.87.5.1937
Free PMC Article

Abstract

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.

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