Amanda Hanks ACF Abstract FY10

Expression, Purification and Characterization of the Asn152Thr Mutant P99 Cephalosporinase

Conference Name: Spring 2010/239th National Meeting of the American Chemical Society

Resistance to β-lactam antibiotics has emerged as a major public health issue due to the over-prescription and widespread use of these drugs. β-lactamase enzymes are the most common bacterial resistance mechanism to these antibiotics and a major concern is evolution of extended β-lactamase enzymes. Several mutants of the class C β-lactamase P99 have been identified that exhibit a substrate selectivity switch due to a mutation of the conserved Asn152 residue. The Asn152Thr mutant was expressed and purified using nickel affinity column chromatography, and pure protein was reproducibly obtained at a concentration of 5 mg/mL.  Crystallization attempts are underway. Structural determination of this extended spectrum enzyme may aid in the design of more effective β-lactam antibiotics.