Expression of cDNA clones by coupled in vitro transcription/translation and transfection into COS-1 cells: protein mapping in two-dimensional gels

Abstract

We present two procedures that can be used to map proteins in two-dimensional gels if the cDNA is at hand. The first procedure, which is illustrated with the expression of cDNAs encoding the fatty acid binding protein 5 (FABP 5), psoriasin and stratifin, makes use of the in vitro transcription/translation assay marketed by Promega. The procedure is simple and allows the mapping of the primary translation product in a very short time. The second method--which faithfully reproduces post-translational modifications--is based on the expression of cDNAs transfected into COS-1 cells using a eukaryotic expression plasmid. This procedure is illustrated with the expression of cDNAs encoding Ha-ras p21 and rab 11, two small GTP-binding proteins known to undergo complex modifications.