Linked Life Data

9636312

Source:http://linkedlifedata.com/resource/pubmed/id/9636312

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1998-7-17
pubmed:abstractText
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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
B
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0733-222X
pubmed:author
pubmed:issnType
Print
pubmed:volume
14
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
50-5
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1996
pubmed:articleTitle
Homologous expression and purification of mutants of an essential protein by reverse epitope-tagging.
pubmed:affiliation
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.