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 cytome galovirus (CMV) immediate-early promoter or the bacteriophage T7 RNA p olymerase 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 p eptide. This peptide consists of a metal ion-binding site, (His)(6), f or single-step affinity purification using Ni2+-chelating resin and a multi-purpose HIV-1-derived peptide (p18(HIV)). This viral epitope can be used to identify, detect and characterize target fusion proteins i n conjunction with a specific monoclonal antibody H902 that does not d isplay cross-reactivity with cellular proteins. The H902 production hy bridoma cell line is reagent #521 from the NIH AIDS Research and Refer ence Program.