Purification of penicillin‐binding protein 4 of Escherichia coli as a soluble protein by dye‐affinity chromatography

The dac B 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°C in E. coli led to the maximum over‐production of PBP4 to 3.8° of the total soluble protein. Expression at 42°C but not at 40°C or 37°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 β‐lactam derivatives did not abolish binding to the dye material, nor was PBP4 eluted by addition of β‐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.