A CLONING AND EPSILON-EPITOPE-TAGGING INSERT FOR THE EXPRESSION OF POLYMERASE CHAIN REACTION-GENERATED CDNA FRAGMENTS IN ESCHERICHIA-COLI AND MAMMALIAN-CELLS

An intercompatible gene-tagging insert sequence was designed to conven iently introduce epitope-tagged polypeptides into bacteria and mammali an cells. The presence of rare restriction enzyme sites located betwee n the ATG codon and the sequence encoding the introduced epsilon-tag c reates a general cloning site which allows efficient cloning of virtua lly any desired cDNA fragment produced by the polymerase chain reactio n (PCR). The tagging insert sequence encodes a KGF-SYFGEDLMP peptide, derived from the last 12 amino acids of the protein kinase C epsilon g ene, to serve as a C-terminal epitope tag of the expressed protein. Wh ile the insert can be readily adapted for insertion into any expressio n vector, this paper details the introduction and characterization of the epsilon-epitope-tagging insert into the bacterial pTrcHis A (epsil on TrcHis A) vector and into the metallothionein promoter-driven eukar yotic (epsilon MTH) expression vector. The expressed epsilon-tagged pr oteins can be readily detected with a commercially available antibody specific for the epsilon-peptide. Immunoscreening of Escherichia coli colonies transformed with the PCR-generated cDNA inserted into the eps ilon TrcHis A vector enables rapid, direct biochemical characterizatio n of the PCR product. The biochemically characterized gene constructs from the epsilon TrcHis A plasmid can be inserted into the epsilon MTH vector by a single subcloning step using the introduced compatible co hesive ends. This epsilon-epitope-tagging insert provides investigator s with a versatile, uncomplicated, and reliable method of expressing a n epitope-tagged PCR product in the cell type of interest. The epsilon -epitope tagging of the expressed peptide facilitated: (1) immunoscree ning of NIH 3T3 transfectants by Western blot analysis for the introdu ced polypeptide, (2) immunoprecipitation of the overexpressed gene pro ducts following metabolic labeling with [S-35]methionine and [P-32]ort hophosphate, and (3) subcellular localization of different recombinant proteins by immunocytochemistry. (C) 1994 Academic Press,Inc.