| pubmed-article:9636312 | rdf:type | pubmed:Citation | lld:pubmed |
| pubmed-article:9636312 | lifeskim:mentions | umls-concept:C0033684 | lld:lifeskim |
| pubmed-article:9636312 | lifeskim:mentions | umls-concept:C0596988 | lld:lifeskim |
| pubmed-article:9636312 | lifeskim:mentions | umls-concept:C0017262 | lld:lifeskim |
| pubmed-article:9636312 | lifeskim:mentions | umls-concept:C1555029 | lld:lifeskim |
| pubmed-article:9636312 | lifeskim:mentions | umls-concept:C1998793 | lld:lifeskim |
| pubmed-article:9636312 | lifeskim:mentions | umls-concept:C0205224 | lld:lifeskim |
| pubmed-article:9636312 | lifeskim:mentions | umls-concept:C1334043 | lld:lifeskim |
| pubmed-article:9636312 | lifeskim:mentions | umls-concept:C2911684 | lld:lifeskim |
| pubmed-article:9636312 | lifeskim:mentions | umls-concept:C0185117 | lld:lifeskim |
| pubmed-article:9636312 | pubmed:issue | 1 | lld:pubmed |
| pubmed-article:9636312 | pubmed:dateCreated | 1998-7-17 | lld:pubmed |
| pubmed-article:9636312 | 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. | lld:pubmed |
| pubmed-article:9636312 | pubmed:language | eng | lld:pubmed |
| pubmed-article:9636312 | pubmed:journal | http://linkedlifedata.com/r... | lld:pubmed |
| pubmed-article:9636312 | pubmed:citationSubset | B | lld:pubmed |
| pubmed-article:9636312 | pubmed:chemical | http://linkedlifedata.com/r... | lld:pubmed |
| pubmed-article:9636312 | pubmed:chemical | http://linkedlifedata.com/r... | lld:pubmed |
| pubmed-article:9636312 | pubmed:chemical | http://linkedlifedata.com/r... | lld:pubmed |
| pubmed-article:9636312 | pubmed:chemical | http://linkedlifedata.com/r... | lld:pubmed |
| pubmed-article:9636312 | pubmed:status | MEDLINE | lld:pubmed |
| pubmed-article:9636312 | pubmed:month | Jan | lld:pubmed |
| pubmed-article:9636312 | pubmed:issn | 0733-222X | lld:pubmed |
| pubmed-article:9636312 | pubmed:author | pubmed-author:SöllDD | lld:pubmed |
| pubmed-article:9636312 | pubmed:author | pubmed-author:HiteA FAF | lld:pubmed |
| pubmed-article:9636312 | pubmed:author | pubmed-author:HongK WKW | lld:pubmed |
| pubmed-article:9636312 | pubmed:author | pubmed-author:ThomannH UHU | lld:pubmed |
| pubmed-article:9636312 | pubmed:issnType | Print | lld:pubmed |
| pubmed-article:9636312 | pubmed:volume | 14 | lld:pubmed |
| pubmed-article:9636312 | pubmed:owner | NLM | lld:pubmed |
| pubmed-article:9636312 | pubmed:authorsComplete | Y | lld:pubmed |
| pubmed-article:9636312 | pubmed:pagination | 50-5 | lld:pubmed |
| pubmed-article:9636312 | pubmed:dateRevised | 2006-11-15 | lld:pubmed |
| pubmed-article:9636312 | pubmed:meshHeading | pubmed-meshheading:9636312-... | lld:pubmed |
| pubmed-article:9636312 | pubmed:meshHeading | pubmed-meshheading:9636312-... | lld:pubmed |
| pubmed-article:9636312 | pubmed:meshHeading | pubmed-meshheading:9636312-... | lld:pubmed |
| pubmed-article:9636312 | pubmed:meshHeading | pubmed-meshheading:9636312-... | lld:pubmed |
| pubmed-article:9636312 | pubmed:meshHeading | pubmed-meshheading:9636312-... | lld:pubmed |
| pubmed-article:9636312 | pubmed:meshHeading | pubmed-meshheading:9636312-... | lld:pubmed |
| pubmed-article:9636312 | pubmed:meshHeading | pubmed-meshheading:9636312-... | lld:pubmed |
| pubmed-article:9636312 | pubmed:meshHeading | pubmed-meshheading:9636312-... | lld:pubmed |
| pubmed-article:9636312 | pubmed:meshHeading | pubmed-meshheading:9636312-... | lld:pubmed |
| pubmed-article:9636312 | pubmed:meshHeading | pubmed-meshheading:9636312-... | lld:pubmed |
| pubmed-article:9636312 | pubmed:meshHeading | pubmed-meshheading:9636312-... | lld:pubmed |
| pubmed-article:9636312 | pubmed:year | 1996 | lld:pubmed |
| pubmed-article:9636312 | pubmed:articleTitle | Homologous expression and purification of mutants of an essential protein by reverse epitope-tagging. | lld:pubmed |
| pubmed-article:9636312 | pubmed:affiliation | Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511, USA. | lld:pubmed |
| pubmed-article:9636312 | pubmed:publicationType | Journal Article | lld:pubmed |
| pubmed-article:9636312 | pubmed:publicationType | Research Support, U.S. Gov't, P.H.S. | lld:pubmed |
| entrez-gene:945310 | entrezgene:pubmed | pubmed-article:9636312 | lld:entrezgene |
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