Expression, Purification And Crystallization Of SrtA And Inhibitors Screening

Staphylococcus aureus(S. aureus) is a Gram-positive bacterium that colonizes human skin and mucosal surfaces. If sensitive to antibiotics, S. aureus infections can be effectively treated withβ-lactam antibiotics, macrolides, and fluoroquinolones. In recent years, with the widespread, sometimes inappropriate and continual use of antimicrobials, more and more people have concerned about the resistance of antibiotic.Bacterial surface proteins function as virulence factors that enable pathogens to adhere to sites of infection, evade the immune response, acquire essential nutrients, and enter host cells. Gram-positive bacteria use a common mechanism to covalently attach proteins to the cell wall. This process is catalyzed by sortase transpeptidase enzymes,which join proteins bearing a highly conserved Leu-Pro-X-Thr-Gly(LPXTG, where X is any amino acid) sorting signal to the cross-bridge peptide of the peptidylglycan. The search for small molecule sortase inhibitors is an active area of research, since these enzymes contribute to the virulence of a number of important pathogens, including among others Staphylococcus aureus, Listeria monocytogenes, Streptococcus pyogenes, and Streptococcus pneumoniae.Sortase A (SrtA) is a polypeptide of 206 amino acids with an N-terminal membrane-spanning region and a C-terminal catalytic domain. SrtAΔN59, a recombinant SrtA lacking the N-terminal 59 residues, is fully functional in vitro and catalyzes the cleavage of peptides bearing an LPXTG motif as well as the transpeptidation reaction with pentaglycine substrate. According to published sequence of the SrtA, we designed two pairs of primers for SrtAΔN59 gene and amplified the SrtAΔN59 gene by polymerase chain reaction (PCR). One of the PCR products was digested with BamhI and SalI, and the fragment was inserted into pQE30 at BamhI and SalI sites. The other one was digested with NdeI and XhoI and the fragment was inserted into pET28b at NdeI and XhoI sites. The recombinant enzyme SrtAΔN59 was cloned and expressed in Escherichia coli. At last, we got the the fusion protein which was purified by the AKTA purify. All crystals were grown by the hanging-drop technique with a protein concentration of 50 mg/ml in 50mM Tris-cl, pH 8.0. The crystallization conditions include D4 and D5 of Grid kit.In order to screen for small molecule inhibitors of SrtA we modified a fluorescence resonance energy transfer assay that monitors the SrtA-catalyzed hydrolysis of an internally quenched fluorescent substrate analog (O-Abz-LPATG-Dap(Dnp)-NH2). For the in vitro assay, we used a total volume of 300 ul reaction(buffer:50 mM Tris-HCl, pH 7.5,150mM NaCl, and 5mM CaCl2), 1μM SrtA, 33μM LPXTG and 1-300μM of the inhibitor. Incubate 30 min at 37℃and then tested the fluorescence(320 nm excitation, 420 nm emission). We selected three compounds which were screened for further study because they had the highest inhibitory activity. Their IC50 values against SrtA are from 126.82μM to 192.85μM. This research has laid the foundation for further study of the discovery and structure–activity relationship analysis of SrtA inhibitors.