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 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.
Kroll
D J
DJ
Division of Medical Oncology, University of Colorado Health Sciences Center, Denver 80262.
Abdel-Malek Abdel-Hafiz
H
H
Marcell
T
T
Simpson
S
S
Chen
C Y
CY
Gutierrez-Hartmann
A
A
Lustbader
J W
JW
Hoeffler
J P
JP
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D004274:DNA, Recombinant; D004277:DNA, Single-Stranded / Q000096:biosynthesis / Q000235:genetics; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli; D015870:Gene Expression; D008969:Molecular Sequence Data; D009532:Nickel; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D004274:DNA, Recombinant; D004277:DNA, Single-Stranded; D000939:Epitopes; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D009532:Nickel