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Expression, Purification and Crystallization of SrtA and Inhibitors Screening

Author: MaZuo
Tutor: YangCaiGuang；ZhangYuMei
School: Yangzhou University
Course: Basic Veterinary Science
Keywords: SrtA enzyme Natural Products X-ray crystal structure Inhibitors
CLC: R378
Type: Master's thesis
Year: 2010
Downloads: 103
Quote: 0
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Abstract

Is recognized as the \Usually Staphylococcus aureus infection can lactam, macrolide or quinolone antibiotics to effective treatment. However, due to the overuse of antibiotics lead to Staphylococcus aureus produces a wide range of resistance. The antibiotics crisis has become a global challenge, the study has been to find the novel structure of the new type of antibiotics imminent. Bacterial surface protein with virulent factor is becoming a new candidate target for antibacterial drug research. The idea is to interfere with bacterial invasion of host infection through regulation of bacterial surface proteins and host interactions. SrtA enzyme is a membrane-bound sulfhydryl GGT, and can identify the surface proteins contained in the C-terminal of the conserved amino acid motif LPXTG (X refers to any amino acid), and cleavage of the threonine (T) and glycine (G) between the peptide bond, resulting in the end of the carbonyl group of a threonine, amido bond is formed with the cell walls of five glycine amino group of the cross-linking bridge, so that the surface protein is anchored to the cell wall peptidoglycan. Since this enzyme is widely present in many important human pathogens, such as monocytes increased Listeria, Streptococcus pyogenes, Streptococcus pneumoniae, etc., based on the study of the mechanism of action of GGT and small molecule inhibitors, as solve the problem for Staphylococcus aureus or chemical treatment of a broad spectrum of bacterial infections will provide strong theoretical basis. SrtA enzyme consists of 206 amino acids, its N-terminal portion of a transmembrane region, and the C-end part of a catalytic region. SrtAΔN59 function in vitro with SrtA same, and can the LPXTG occur with substrate turn peptide reactive, catalytic T and G between the fracture. SrtA gene sequence according to known, we designed a primer the two pairs SrtAΔN59 gene sequence, was amplified by polymerase chain reaction (PCR). Wherein a PCR product by the restriction endonucleases BamHI and SalI double enzyme is then subcloned into the vector pQE30, another PCR product was cut by the restriction enzyme NdeI and XhoI double enzymes, then subcloned into the vector pET28b. Expressed in E. coli the the recombinant protein SrtAΔN59, and using the AKTA purification device was then purified to obtain a purity higher than 90% of the SrtAΔN59 protein. Concentrated to the purified after SrtAΔN59 protein to 50mg/ml, using the hanging drop method screening crystals obtained SrtA the crystal, D4 and D5 of the kit in the Grid. Filter SrtA small molecule inhibitors of the enzyme, we utilize a fluorescence resonance energy transfer principle, synthetic SrtA enzyme substrate (O-aminobenzoyl-LPATG-diaminopropionic acid-dinitrophenyl-NH2, of ABZ-LPATG-Dap (Dnp)-NH2) . When substrates LPATG SrtA enzyme is not cut off, the weak absorption of certain wavelengths of fluorescence, but when it is SrtA digested fractured, the fluorescence at this wavelength is absorbed will be greatly enhanced. In the process of screening inhibitors SrtA by monitoring enzyme inhibition the fluorescence absorption values ??after the reaction to determine the activity of small molecule compounds. Finally, we screened the three has a strong inhibitory activity, their IC50 values ??126.82μM, 138.26μM and 192.85μM respectively. The study laid a foundation for the future SrtA inhibitors discovery and analysis of the structure-activity relationship between them.

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