A Multifunctional Prokaryotic Protein Expression System: Overproduction, Affinity Purification, and Selective Detection

Abstract

A series of plasmid vectors, pRSET A, B, and C, have been developed for high-level protein expression in prokaryotes and have been characterized. Based upon the T7 RNA polymerase-driven pET system, the pRSET vectors encode recombinant proteins as fusions with a multifunctional leader peptide containing a hexahistidyl sequence for purification on Ni(2+)-affinity resins, a tyrosine residue for radioiodination, and an enterokinase proteolytic cleavage site for leader peptide removal. Monoclonal antibodies (MAbs) to two epitopes on the leader peptide, which also contains amino acids 1-12 of the T7 gene 10 major capsid protein, were developed and provide for universal immunological detection of pRSET-expressed fusion proteins. Subcloning of protein-encoding DNA is facilitated by an 11-site polylinker which is offset for all three ribosomal reading frames, and an f1(+) origin of DNA replication permits single-stranded DNA synthesis for site-directed mutagenesis protocols. Representative fusion proteins overexpressed in Escherichia coli were successfully purified under both denaturing and nondenaturing conditions by single-step Ni2+ affinity chromatography. Purification was independent 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 immunoprecipitation, but this selectivity was less evident under conditions for Western blotting. In combining the utility of T7 RNA polymerase-directed expression with several recent advances in protein purification and detection, the pRSET vectors will serve as a powerful resource for a variety of studies in protein biochemistry.

TAF9b (Formerly TAF9L) Is a Bona Fide TAF That Has Unique and Overlapping Roles with TAF9

“…Yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) TAF9b fusions were generated by subcloning TAF9b cDNA from pXJ41 using BamHI and EcoRI in BglII and EcoRI sites of pEYFP-C1 and pECFP-C1 (Clontech), respectively. Subcloning the NheI excised and blunted TAF6 and TAF9 cDNA fragments from pRSET vector (31) into the blunted BamHI in EcoRI sites of pEYFP-C1 and pECFP-C1 vectors, respectively, generated YFP-and CFP-TAF9 as well as YFP-and CFP-TAF6 fusions.…”

“…TAF9 was first identified as a TFIID subunit from multiple organisms: human (formerly called hTAF II 31 or hTAF II 32 [29,35]), Drosophila melanogaster (formerly dTAF II 40 [55]), and yeast (formerly yTaf17p [42]). Later, TAF9 was also identified as a component of different TBP-free TAF complexes containing the GCN5-type histone acetyltransferase, such as the yeast SAGA complex (19), the Drosophila TFTC complex (44), and human TFTC-type complexes (10,36,67).…”

Segregation of B and T Cell Epitopes of Treponema pallidum Repeat Protein K to Variable and Conserved Regions During Experimental Syphilis Infection

“…The amplicons from T. pallidum Seattle Nichols strain DNA were cloned into the pRSET expression vector (Invitrogen, Carlsbad, CA), the sequence was verified, and the peptide was expressed in Escherichia coli and purified as previously described (7,13). The proteins were dialyzed into PBS, pH 7.2, size confirmation and purity were evaluated by SDS-PAGE, and concentrations were determined using a bicinchoninic acid protein assay (Pierce, Rockford, IL).…”

The role of cysteine residues in structure and enzyme activity of a maize β‐glucosidase

“…The expression vector pRSET A [14] was purchased from Invitrogen BV (San Diego, CA, USA). Restriction enzymes, T4-DNA ligase and Taq polymerase were obtained from Boehringer Mannheim GmbH (Mannheim, Germany).…”