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Title: β-lactamase of Staphylococcus aureus PC1 : studies using site-directed mutagenesis
Author: Smith, Thomas John
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1992
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A system was developed for generating site-directed mutants of the β-lactamase of Staphylococcus aureus PC1, and for expression of the mutant proteins in S.aureus. The mutant genes were made in vitro using the polymerase catalysed chain reaction (PCR), by the overlap extension method (Ho, S.N. et al. [1989] Gene 77, 51-59). The were cloned in Escherichia coli and transferred to S.aureus using an E.coli-S.aureus shuttle vector. The mutant proteins were, like the wild-type, secreted into the medium by the transformed S.aureus cells. It was found that high-level constitutive expression, at a level that was possible for the wild-type-β-lactamase, was not obtainable with the three mutants that were studied in this work. Mutations of the β-lactamase Shine-Dalgarno sequence, which originally occurred spontaneously, were exploited to enable the mutant genes to be established in S.aureus at a reduced level of expression. The S70A mutation (amino acid numbering is according to Ambler, R.P. et al. [1991] Biochemical Journal 276, 269-270) was designed to abolish catalysis whilst preserving binding of the substrate. My co-workers have crystallised the native mutant enzyme and obtained a high-resolution electron density map. Work is under way to obtain similar data for complexes between the mutant enzyme and substrates or inhibitors. The S130A mutant has diminished activity, but k_cat for ampicillin and benzylpenicillin is affected considerably more than for cephaloridine and nitrocefin. The steady-state kinetic properties of the A238S mutant are not greatly altered, although the mutant is more strongly inhibited by cefotaxime than the wild-type is. It was observed that a 0.84kb HindIII-XbaI fragment, containing part of the staphylococcal β-lactamase gene, is unstable in E.coli when cloned in silation from the rest of the gene. All the clones that were characterised had undergone changes that would reduce expression of the truncated β-lactamase gene; a large proportion of these had mutations of the promoter region. It is proposed that expression of the truncated gene is a strong selective disadvantage to E.coli, although the intact gene is essentially harmless.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available