Rapid mutagenesis and purification of phage RNA polymerases.
We have developed plasmid-based expression systems that encode modified forms of T7 RNA polymerase (RNAP) having 6-12 histidine residues fused to the amino terminus. The histidine-tagged RNAPs (His-T7 RNAPS) are indistinguishable from the wild-type (WT) enzyme in nearly all biochemical assays. Similar plasmids that encode His-tagged T3 and SP6 RNAPs have also been constructed. To facilitate site-directed mutagenesis of the RNAP gene, the size of the target plasmid was minimized by using T7 RNAP itself as a selectable marker. BL21 (DCAT4) cells (which carry a chromosomal copy of the chloramphenicol acetyltransferase cat gene under control of a T7 promoter) are resistant to chloramphenicol when functional T7 RNAP is expressed, thus allowing the selection and maintenance of the target plasmid in these cells. Mutagenesis is accomplished by denaturing the plasmid, annealing mutagenic DNA primers, and repairing the plasmid with T4 DNA polymerase. Two DNA primers are used: one corrects a defect in the bla gene, the other introduces the desired mutation into the RNAP gene; 30-85% of the ampicillin-resistant transformants carry the desired mutation in the RNAP gene. By using BL21 (DCAT4) cells as a recipient for transformation the functional integrity of the RNAP gene may conveniently be monitored by assessing the level of chloramphenicol resistance in vivo. Methods for rapid, simultaneous purification of multiple samples of modified (His-tagged) and conventional RNAPs are described. Together, these developments greatly enhance our ability to characterize this important class of enzymes.
He
B
B
Department of Microbiology and Immunology, State University of New York, Health Science Center at Brooklyn, 11203-2098, USA.
Rong
M
M
Lyakhov
D
D
Gartenstein
H
H
Diaz
G
G
Castagna
R
R
McAllister
W T
WT
Durbin
R K
RK
D000595:Amino Acid Sequence; D017903:Bacteriophage T3 / Q000201:enzymology / Q000235:genetics; D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase; D002846:Chromatography, Affinity; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000302:isolation & purification*; D006639:Histidine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012641:Selection, Genetic
D010455:Peptides; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D006639:Histidine; D015500:Chloramphenicol O-Acetyltransferase; D012321:DNA-Directed RNA Polymerases