Homologous Expression and Purification of Mutants of an Essential Protein by Reverse Epitope-Tagging

Abstract

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.

Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins in vivo

“…A purification of GlnRS mutants free of endogenous wt GlnRS has recently been developed using a strain of E. coli (HAPPY101), in which a hemagglutinin epitope has been attached to the genomic copy of the glutaminyl-tRNA aminoacyl synthetase gene (5). Using this method, several mutant synthetases surviving each round of selection were purified and assayed in vitro for their ability to acylate wt tRNA Gln (as a mixture of all E. coli tRNAs including tRNA 1 Gln and tRNA 2 Gln ) and the O-tRNA.…”

“…Mutant enzymes were purified by anion exchange chromatography on a Mono Q HR 10͞10 fast protein liquid chromatography column by adapting published protocols (5). To analyze each fraction for the presence of hemagglutinin (HA)-tagged wt GlnRS, aliquots of each column fraction were incubated in a 96-well plate and subjected to ELISA using 12CA5 mouse anti-HA IgG solution (Boehringer Mannheim, 1.0 g͞ml) and goat anti-mouse antibodies conjugated to alkaline phosphatase (Sigma, 2 g͞ml).…”

Interactions between tRNA identity nucleotides and their recognition sites in glutaminyl-tRNA synthetase determine the cognate amino acid affinity of the enzyme.

“…The E. coli strain BL21(DE3) (28) was used for the overexpression of wild-type GlnRS. GlnRS-R402A was overexpressed in strain HAPPY101 (29) and the variants K317R and Q318K were overexpressed in the glnS deletion strain X3R2 (30).…”

“…Wild-type, K317R, Q318K, and R402A GlnRS were purified as described (29) except that immunodetection of HA-GlnRS was omitted for the first three. Aminoacylation Assays.…”

Retracing the evolution of amino acid specificity in glutaminyl‐tRNA synthetase

“…Wild‐type [9]and mutant [10]GlnRS proteins, as well as dihydrofolate reductase (DHFR [11]), were produced and purified as previously described. tRNA Gln 2 was a gift from J.M.…”