Oct 1, 1991

Expression and purification of a soluble form of penicillin-binding protein 2 from both penicillin-susceptible and penicillin-resistant Neisseria gonorrhoeae

Protein Expression and Purification
D E SchultzRobert A Nicholas

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 induce...Continue Reading

Mentioned in this Paper

Monobactams
Shuttle Vectors
Bacterial Proteins
Tissue Membrane
Alkalescens-Dispar Group
Enzymes, antithrombotic
expression vector
Muramoylpentapeptide Carboxypeptidase
Penicillin Resistance
Peptidoglycan synthetase

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