ENGINEERING AND OVEREXPRESSION OF PERIPLASMIC FORMS OF THE PENICILLIN-BINDING PROTEIN-3 OF ESCHERICHIA-COLI

Replacement of the 36 and 56 N-terminal amino acid residues of the 588 -amino-acid-residue membrane-bound penicillin-binding protein 3 (PBP3) of Escherichia coli by the OmpA signal peptide allows export of F37-V 577 PBP3 and G57-V577 PBP3 respectively into the periplasm. The modifi ed ftsI genes were placed under the control of the fused lpp promoter and lac promoter/operator; expression of the truncated PBP3s was optim ized by varying the copy number of the recombinant plasmids and the am ount of LacI repressor, and export was facilitated by increasing the S ecB content of the producing strain. The periplasmic PBP3s (yield 8 mg /l of culture) were purified to 70 % protein homogeneity. They require the presence of 0.25 M NaCl to remain soluble. Like the membrane-boun d PBP3, they undergo processing by elimination of the C-terminal decap eptide I578-S588, they bind penicillin in a 1:1 molar ratio and they c atalyse hydrolysis and aminolysis of acyclic thioesters that are analo gues of penicillin. The membrane-anchor-free PBP3s have ragged N-termi ni. The G57-V577 PBP3, however, is less prone to proteolytic degradati on than the F37-V577 PBP3.