HOMOLOGOUS EXPRESSION AND PURIFICATION OF MUTANTS OF AN ESSENTIAL PROTEIN BY REVERSE EPITOPE-TAGGING

Purification of mutant enzymes is a prime requirement of biophysical a nd biochemical studies. Our investigations on the essential Escherichi a coli enzyme glutaminyl-tRNA synthetase demand mutant enzymes free of any wild-type protein contamination. However, as it is not possible t o express noncomplementing mutant enzymes in an E. coli glnS-deletion strain, we developed a novel strategy to address these problems. Inste ad of following the common tactic of epitope-tagging the mutant protei n of interest on an extrachromosomal genetic element, we fused a repor ter epitope to the 5' end of the chromosomal glnS-gene copy: this is r eferred to as 'reverse epitope-tagging.' The corresponding strain, E. coli HAPPY101, displays a normal phenotype, and glutaminyl-tRNA synthe tase is exclusively present as an epitope-tagged form in cell-free ext racts, Here we report the use of E. coli HAPPY101 to express and purif y a number of mutant glutaminyl-tRNA synthetases independently of thei r enzymatic activity. In this process, epitope-tagged wild-type protei n is readily separated from mutant enzymes by conventional chromatogra phic methods. In addition, the absence of wild-type can be monitored b y immunodetection using a monoclonal antibody specific for the epitope . The strategy described here for expression and purification of an es sential enzyme is not restricted to glutaminyl-tRNA synthetase and sho uld be applicable to any essential enzyme that retains sufficient acti vity to sustain growth following reverse epitope-tagging.