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

By Luis C Ferreira, Uli Schwarz, Wolfgang Keck, Paulette Charlier, Otto Dideberg and Jean-Marie Ghuysen

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. PBP5S). In PBP5S the carboxyl-terminal 21-amino-acid region of the wild-type protein was replaced by a short 9-amino-acid segment. Milligram amounts of PBP5S were purified by penicillin affinity chromatography in the absence of detergents or of chaotropic agents. PBP5S 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 PBP5S were crystallized. In contrast to PBP5, PBP5S yielded enzymatically active, well-formed prismatic crystals suitable for X-ray analysis.Peer reviewe

Topics: bacterial proteins, carrier proteins/genetics, crystallization, escherichia coli/enzymology/*genetics, genetic engineering, hexosyltransferases, molecular weight, muramoylpentapeptide carboxypeptidase/genetics, penicillin-binding proteins, peptidyl transferases, serine endopeptidases/genetics, solubility, Life sciences :: Biochemistry, biophysics & molecular biology, Sciences du vivant :: Biochimie, biophysique & biologie mol├ęculaire
Year: 1988
OAI identifier: oai:orbi.ulg.ac.be:2268/81280
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