Properties and crystallization of a genetically engineered, water‐soluble derivative of penicillin‐binding protein 5 of Escherichia coli K12

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Abstract
Derivatives of the Escherichia coli penicillin-binding protein 5 (PBP5) with truncated carboxyl terminals were obtained by altering the carboxyl-coding end of the dacA gene. After cloning the modified dacA gene into a runaway-replication-control plasmid, one clone that overproduced and excreted the desired protein into the periplasm was used as a source for the isolation of a water-soluble PBP5 (i.e. PBP55). In PBP55 the carboxyl-terminal 21-amino-acid region of the wild-type protein was replaced by a short 9-amino-acid segment. Milligram amounts of PBP55 were purified by pencillin affinity chromatography in the absence of detergents or of chaotropic agents. PBP55 was stable and possessed DD-carboxypeptidase activity without added Triton X-100. Upon reaction with [14C]benzylpenicillin it was converted into a rather short-lived acyl-enzyme complex, as observed with PBP5. Both PBP5 and PBP55 were crystallized. In contrast to PBP5, PBP55 yielded enzymatically active, well-formed prismatic crystals suitable for X-ray analysis.