An efficient expression, purification and immunodetection system for recombinant gene products.

We describe a modification of the mammalian expression vector pRc/CMV, which drives expression of inserted genes from either the human cytomegalovirus (CMV) immediate-early promoter or the bacteriophage T7 RNA polymerase promoter. The modification is designed to allow expression, simple purification and specific immunodetection of recombinant fusion proteins. The modified plasmid, termed pTag/CMV-neo, encodes a Kozak consensus ribosome-binding site (RBS) and a 30-amino acid fusion tag peptide. This peptide consists of a metal ion-binding site, (His)6, for single-step affinity purification using Ni(2+)-chelating resin and a multi-purpose HIV-1-derived peptide (p18HIV). This viral epitope can be used to identify, detect and characterize target fusion proteins in conjunction with a specific monoclonal antibody H902 that does not display cross-reactivity with cellular proteins. The H902 production hybridoma cell line is reagent #521 from the NIH AIDS Research and Reference Program.