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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
3
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pubmed:dateCreated |
1991-11-1
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pubmed:abstractText |
The dacB gene of Escherichia coli, coding for penicillin-binding protein 4 (PBP4) was cloned under the control of the phage lambda pR promoter and cro gene translation signals. Depression of the phage lambda promoter for 2 h at 42 degrees C in E. coli led to the maximum over-production of PBP4 to 3.8% of the total soluble protein. Expression at 42 degrees C but not at 40 degrees C or 37 degrees C led to incomplete processing and aggregation of the preform of PBP4. Cibacron navyblue 2G-E was selected from a collection of triazine dyes as having a high affinity for PBP4. The immobilised dye was used in a two-step procedure to isolated 374 mg PBP4 from the soluble fraction of 125 g (wet mass) cells of the over-producing strain, with a recovery of 63.2% and a final purity of 99% as determined by active-site titration with radiolabelled penicillin. Saturation of PBP4 with various beta-lactam derivatives did not abolish binding to the dye material, nor was PBP4 eluted by addition of beta-lactams from the dye matrix. PBP4 behaved as a soluble protein throughout the purification, that was performed in the complete absence of detergents. Furthermore, in flotation experiments on sucrose density gradients and in Triton X-114 fractionation experiments, it showed the characteristics of a soluble protein. Cibacron navyblue 2G-E showed class specificity for all E. coli PBP except PBP3 and could be used for the isolation of these PBP from membrane extracts.
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/Bacterial Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Carrier Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Coloring Agents,
http://linkedlifedata.com/resource/pubmed/chemical/Escherichia coli Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Hexosyltransferases,
http://linkedlifedata.com/resource/pubmed/chemical/Muramoylpentapeptide...,
http://linkedlifedata.com/resource/pubmed/chemical/Penicillin-Binding Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Peptidyl Transferases,
http://linkedlifedata.com/resource/pubmed/chemical/Serine-Type D-Ala-D-Ala...,
http://linkedlifedata.com/resource/pubmed/chemical/Triazines,
http://linkedlifedata.com/resource/pubmed/chemical/penicillin-binding protein 4, E coli
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pubmed:status |
MEDLINE
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pubmed:month |
Sep
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pubmed:issn |
0014-2956
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pubmed:author |
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pubmed:issnType |
Print
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pubmed:day |
15
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pubmed:volume |
200
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
767-73
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pubmed:dateRevised |
2008-11-21
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pubmed:meshHeading |
pubmed-meshheading:1833192-Bacterial Proteins,
pubmed-meshheading:1833192-Bacteriophage lambda,
pubmed-meshheading:1833192-Base Sequence,
pubmed-meshheading:1833192-Carrier Proteins,
pubmed-meshheading:1833192-Centrifugation, Density Gradient,
pubmed-meshheading:1833192-Chromatography, Affinity,
pubmed-meshheading:1833192-Cloning, Molecular,
pubmed-meshheading:1833192-Coloring Agents,
pubmed-meshheading:1833192-Escherichia coli,
pubmed-meshheading:1833192-Escherichia coli Proteins,
pubmed-meshheading:1833192-Gene Expression,
pubmed-meshheading:1833192-Hexosyltransferases,
pubmed-meshheading:1833192-Molecular Sequence Data,
pubmed-meshheading:1833192-Muramoylpentapeptide Carboxypeptidase,
pubmed-meshheading:1833192-Penicillin-Binding Proteins,
pubmed-meshheading:1833192-Peptidyl Transferases,
pubmed-meshheading:1833192-Plasmids,
pubmed-meshheading:1833192-Promoter Regions, Genetic,
pubmed-meshheading:1833192-Serine-Type D-Ala-D-Ala Carboxypeptidase,
pubmed-meshheading:1833192-Solubility,
pubmed-meshheading:1833192-Triazines
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pubmed:year |
1991
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pubmed:articleTitle |
Purification of penicillin-binding protein 4 of Escherichia coli as a soluble protein by dye-affinity chromatography.
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pubmed:affiliation |
University of Groningen, Department of Biochemistry, The Netherlands.
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pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
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