Expression-PCR (E-PCR): overview and applications.

1Department of Immunology, Waiter Reed Army Institute of Research, Washington, D.C. 20307-5100; 2Tropical Disease Unit, Division of Infectious Diseases, The Toronto Hospital and The University of Toronto, Toronto, Ontario, Canada M5G 2C4 In vitro transcription and translation are powerful tools for examining the s t ructure-funct ion relationships of proteins. Genes can be cloned into plasmid vectors containing the bacteriophage promoters T7, T3, and SP6 and then transcribed in the presence of an appropriate RNA polymerase. However, efficient in vitro translation of these RNA transcripts often requires the insertion of an appropriate untranslated leader sequence downstream from the promoter to provide a suitable context for ribosomal binding and init iation of protein synthesis. Standard methods for in vitro transcript ion and translation are limited further by their requirements for cloning, bacterial amplification, DNA extraction, and restriction enzyme digestion before the desired DNA template can be transcribed and translated. We believe that it would be of great advantage to express functional proteins from DNA without these constraints. Because of the ease of obtaining adequate quantities of any gene segment by the use of PCR, we designed a method called expression-PCR (E-PCR) (1) to modify this DNA so that it could be expressed without having to go through the rigors of cloning. E-PCR is a rapid, simple method for the in vitro product ion of proteins wi thout cloning. The resulting radiochemically pure proteins are useful for a variety of purposes, including studies on the subunit structure of proteins, epitope mapping, and protein mutagenesis.