Homologous expression and purification of mutants of an essential protein by reverse epitope-tagging.
Purification of mutant enzymes is a prime requirement of biophysical and biochemical studies. Our investigations on the essential Escherichia coli enzyme glutaminyl-tRNA synthetase demand mutant enzymes free of any wild-type protein contamination. However, as it is not possible to express noncomplementing mutant enzymes in an E. coli glnS-deletion strain, we developed a novel strategy to address these problems. Instead of following the common tactic of epitope-tagging the mutant protein of interest on an extrachromosomal genetic element, we fused a reporter epitope to the 5' end of the chromosomal glnS-gene copy: this is referred to as 'reverse epitope-tagging.' The corresponding strain, E. coli HAPPY101, displays a normal phenotype, and glutaminyl-tRNA synthetase is exclusively present as an epitope-tagged form in cell-free extracts. Here we report the use of E. coli HAPPY101 to express and purify a number of mutant glutaminyl-tRNA synthetases independently of their enzymatic activity. In this process, epitope-tagged wild-type protein is readily separated from mutant enzymes by conventional chromatographic methods. In addition, the absence of wild-type can be monitored by immunodetection using a monoclonal antibody specific for the epitope. The strategy described here for expression and purification of an essential enzyme is not restricted to glutaminyl-tRNA synthetase and should be applicable to any essential enzyme that retains sufficient activity to sustain growth following reverse epitope-tagging.
Thomann
H U
HU
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511, USA.
Ibba
M
M
Hong
K W
KW
Söll
D
D
D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics* / Q000302:isolation & purification; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D019677:Artificial Gene Fusion; D002872:Chromosome Deletion; D000939:Epitopes / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid
D000911:Antibodies, Monoclonal; D000939:Epitopes; D000604:Amino Acyl-tRNA Synthetases; C020659:glutaminyl-tRNA synthetase