PURIFICATION OF PENICILLIN-BINDING PROTEIN-4 OF ESCHERICHIA-COLI AS A SOLUBLE-PROTEIN BY DYE-AFFINITY CHROMATOGRAPHY

The dacB gene of Escherichia coli, coding for penicillin-binding protein 4 (PBP4) was cloned under the control of the phage lambda-p(R) promoter and cro gene translation signals. Derepression 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.