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

Bio/Technology volume 14, pages 50–55 (1996)Cite this article

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 non-complementing 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.

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Author notes

  1. Hans-Ulrich Thomann

    Present address: Genome Therapeutics Corp., 100 Beaver Street, Waltham, MA, 02154

Authors and Affiliations

  1. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, 06511

    Michael Ibba, Kwang-Won Hong & Dieter Söll

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  1. Hans-Ulrich Thomann
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  2. Michael Ibba
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  4. Dieter Söll
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Correspondence to Dieter Söll.

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Thomann, HU., Ibba, M., Hong, KW. et al. Homologous Expression and Purification of Mutants of an Essential Protein by Reverse Epitope-Tagging. Nat Biotechnol 14, 50–55 (1996). https://doi.org/10.1038/nbt0196-50

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  • DOI: https://doi.org/10.1038/nbt0196-50

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