PMID:1833192
| Citation |
Mottl, H and Keck, W (1991) Purification of penicillin-binding protein 4 of Escherichia coli as a soluble protein by dye-affinity chromatography. Eur. J. Biochem. 200:767-73 |
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| Abstract |
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. |
| Links | |
| Keywords |
Bacterial Proteins; Bacteriophage lambda/genetics; Base Sequence; Carrier Proteins/genetics; Carrier Proteins/isolation & purification; Centrifugation, Density Gradient; Chromatography, Affinity; Cloning, Molecular; Coloring Agents; Escherichia coli/genetics; Escherichia coli Proteins; Gene Expression; Hexosyltransferases; Molecular Sequence Data; Muramoylpentapeptide Carboxypeptidase/genetics; Muramoylpentapeptide Carboxypeptidase/isolation & purification; Penicillin-Binding Proteins; Peptidyl Transferases; Plasmids; Promoter Regions, Genetic; Serine-Type D-Ala-D-Ala Carboxypeptidase; Solubility; Triazines |
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