title: Purification of penicillin-binding protein 4 of Escherichia coli as a soluble protein by dye-affinity chromatography.
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.
authors.affiliation: University of Groningen, Department of Biochemistry, The Netherlands.
authors.forename: H
authors.initials: H
authors.lastname: Mottl
authors.forename: W
authors.initials: W
authors.lastname: Keck
mesh_terms: D001426:Bacterial Proteins; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification*; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004396:Coloring Agents; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D006602:Hexosyltransferases; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics / Q000302:isolation & purification*; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D010957:Plasmids; D011401:Promoter Regions, Genetic; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; D012995:Solubility; D014227:Triazines
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D004396:Coloring Agents; D029968:Escherichia coli Proteins; D046915:Penicillin-Binding Proteins; D014227:Triazines; C071073:Cibacron navyblue 2G-E; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; C478706:penicillin-binding protein 4, E coli; D002267:Muramoylpentapeptide Carboxypeptidase