title: High-level expression, purification, and characterization of Staphylococcus aureus dihydroorotase (PyrC) as a cleavable His-SUMO fusion.
abstract: Staphylococcus aureus is a pathogenic bacterium that causes a variety of mild to lethal human diseases. The rapid spread of multidrug-resistant strains makes the discovery of new antimicrobial agents critical. Dihydroorotase (PyrC), the third enzyme in the bacterial pyrimidine biosynthesis pathway, is structurally and mechanistically distinct from its mammalian counterpart. It has been confirmed to be essential in S. aureus making it an attractive antibacterial drug target. No protocol to express and purify S. aureus PyrC (SaPyrC) has been reported. To obtain the SaPyrC enzyme and overcome anticipated solubility problems, the SaPyrC gene was cloned into the pET-SUMO vector. The N-terminal His-SUMO fused SaPyrC was expressed in Escherichia coli BL21 (DE3) with an HRV 3C protease recognition site inserted between the SUMO tag and SaPyrC to allow for improved cleavage by HRV protease. Purification of cleaved protein using HisTrap affinity and gel filtration columns resulted in native SaPyrC with estimated 95% purity and 40% yield. Both His-SUMO tagged and native SaPyrC form dimers, and enzyme characterization studies have shown that the His-SUMO tag affects enzyme activity slightly. Forward and reverse kinetic rate constants for both tagged and native SaPyrC were determined, and pH profiling studies revealed the optimal pH values for forward and reverse reactions.
authors.affiliation: Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, 900 S. Ashland Ave., Suite 3100, Chicago, IL 60607-7173, USA.
authors.forename: Lena
authors.initials: L
authors.lastname: Truong
authors.forename: Kirk E
authors.initials: KE
authors.lastname: Hevener
authors.forename: Amy J
authors.initials: AJ
authors.lastname: Rice
authors.forename: Kavankumar
authors.initials: K
authors.lastname: Patel
authors.forename: Michael E
authors.initials: ME
authors.lastname: Johnson
authors.forename: Hyun
authors.initials: H
authors.lastname: Lee
mesh_terms: D000900:Anti-Bacterial Agents / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004080:Dihydroorotase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism; D013203:Staphylococcal Infections / Q000201:enzymology / Q000235:genetics / Q000382:microbiology; D013211:Staphylococcus aureus / Q000201:enzymology*
chemical_list: D000900:Anti-Bacterial Agents; D011993:Recombinant Fusion Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D004080:Dihydroorotase