title: Expression and purification of a soluble form of penicillin-binding protein 2 from both penicillin-susceptible and penicillin-resistant Neisseria gonorrhoeae.
abstract: Resistance to penicillin in non-beta-lactamase-producing strains of Neisseria gonorrhoeae (CMRNG strains) is mediated in part by the production of altered forms of penicillin-binding protein 2 (PBP 2) that have a decreased affinity for penicillin. The reduction in the affinity of PBP 2 is largely due to the insertion of an aspartic acid residue (Asp-345a) into the amino acid sequence of PBP 2. Truncated forms of N. gonorrhoeae PBP 2, which differed only by the insertion of Asp-345a, were constructed by placing the region of the penA genes encoding the periplasmic domain of PBP 2 (amino acids 42-581) into an ATG expression vector. When the recombinant PBP 2 molecules were overexpressed in Escherichia coli, insoluble PBP 2 inclusion bodies, which could be isolated by low-speed centrifugation of cell lysates, were formed. These insoluble aggregates were solubilized and the truncated PBP 2 polypeptides were partially purified by cation-exchange chromatography and gel filtration in the presence of denaturant prior to the refolding of the enzyme in vitro. After renaturation, gel filtration was used to separate monomeric soluble PBP 2 from improperly folded protein aggregates and other protein contaminants. A 4-liter culture of induced E. coli cells yielded 1.4 mg of soluble PBP 2 or PBP 2' (PBP 2 containing the Asp-345a insertion), both of which were estimated to be 99% pure. The affinity of soluble PBP 2' for [3H]penicillin G was decreased fourfold relative to that of soluble PBP 2, and their affinities were found to be identical to the affinities of the full-length PBP 2 enzymes that were previously determined in N. gonorrhoeae membranes. Furthermore, soluble PBP 2 displayed a rank order of affinity for several other beta-lactam antibiotics that was consistent with the rank order of affinities previously reported for the native molecules. On the basis of these results, both of these soluble PBPs should be suitable for crystallization and X-ray crystallographic analysis.
authors.affiliation: Department of Pharmacology, University of North Carolina, Chapel Hill 27599-7365.
authors.forename: D E
authors.initials: DE
authors.lastname: Schultz
authors.forename: B G
authors.initials: BG
authors.lastname: Spratt
authors.forename: R A
authors.initials: RA
authors.lastname: Nicholas
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002352:Carrier Proteins; D002850:Chromatography, Gel; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006602:Hexosyltransferases / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics / Q000302:isolation & purification*; D002267:Muramoylpentapeptide Carboxypeptidase; D016297:Mutagenesis, Site-Directed; D009344:Neisseria gonorrhoeae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D010403:Penicillin Resistance / Q000235:genetics; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D009097:Multienzyme Complexes; D046915:Penicillin-Binding Proteins; D011994:Recombinant Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase