A MULTIFUNCTIONAL PROKARYOTIC PROTEIN EXPRESSION SYSTEM - OVERPRODUCTION, AFFINITY PURIFICATION, AND SELECTIVE DETECTION

A series of plasmid vectors, pRSET A, B, and C, have been developed fo r high-level protein expression in prokaryotes and have been character ized. Based upon the T7 RNA polymerase-driven pET system, the pRSET ve ctors encode recombinant proteins as fusions with a multifunctional le ader peptide containing a hexahistidyl sequence for purification on Ni 2+-affinity resins, a tyrosine residue for radioiodination, and an ent erokinase proteolytic cleavage site for leader peptide removal. Monocl onal antibodies (MAbs) to two epitopes on the leader peptide, which al so contains amino acids 1-12 of the T7 gene 10 major capsid protein, w ere developed and provide for universal immunological detection of pRS ET-expressed fusion proteins. Subcloning of protein-encoding DNA is fa cilitated by an 11-site polylinker which is offset for all three ribos omal reading frames, and an f1(+) origin of DNA replication permits si ngle-stranded DNA synthesis for site-directed mutagenesis protocols. R epresentative fusion proteins overexpressed in Escherichia coli were s uccessfully purified under both denaturing and nondenaturing condition s by single-step Ni2+ affinity chromatography. Purification was indepe ndent of recombinant protein solubility in sonicated or freeze-thawed E. coli lysates. Isolation of MAbs for selective recognition of either of two leader peptide epitopes was demonstrated by immunoprecipitatio n, but this selectivity was less evident under conditions for Western blotting. In combining the utility of T7 RNA polymerase-directed expre ssion with several recent advances in protein purification and detecti on, the pRSET vectors will serve as a powerful resource for a variety of studies in protein biochemistry.