Crystal Structure And Functional Research On The Surface Protein Sdre Of Staphylococcus Aureus

Objective The SdrE protein, one of the staphylococcus aureus (S.aureus) surface proteins, plays a key role in bone infections. In order todetermine the three dimensional structure, analyze the molecularmechanism on Staphylococcus aureus infect and develop an newantibacterial drug, we cloned, expressed, purified and crystallized thesurface protein SdrE from S. aureus, and we researched SdrEcorresponding functions through the knockout and mutation.Methods SdrE gene was amplified and cloned into the vector. Therecombinant plasmid, pW28-SdrE, was transformed into E. coli B834toexpress SdrE protein. The SdrE protein was purified by Ni2+-NTA affinitychromatography column and DEAE anion-exchange chromatographycolumn, analyzed the state of aggregation in solution by Hiload Superdex200gel flittration column. The crystals of SdrE and Se-SdrE were screenedwith Hampton kit and optimized crystals with chessboard method, and thediffraction data were collected by X-ray on beamline BL17U of theShanghai Synchrotron Radiation Facility (SSRF). Target genes were knocked out and mutated using the overlap extension PCR and allelicreplacement. On this basis, we analyzed relations between SdrE andstaphylococcus aureus resistance through the drug sensitive test, verifiedthe interaction of SdrE and proteins by Pull-down, SPR and ITC, studiedthe relations between SdrE and staphylococcus aureus infection withadhesion and invasion, and further studied the virulence of SdrE byresearching animals.Results We constructed the fusion plasmid pW28-SdrE and puriedSdrE protein which exhibits as a monomer in solution, and we puriedSe-SdrE protein with the purity of85%. The single crystals, including SdrEand Se-SdrE, were obtained, and the diffraction data were collected byX-ray on beamline BL17U of the SSRF. The SdrE belongs to P222spacegroup, with unit-cell parameters a=45.19, b=66.35, c=146.22, and onemolecule is accommodated per asymmetc unit. In addition, we constructedgene deletion strains of SdrE from staphylococcus aureus, and proved SdrEis closely related to the invasion of staphylococcus aureus.Conclusions The SdrE crystals were grown by classic methods ofprotein purification and crystal cultivation, and we verified SdrEcontributed to staphylococcus aureus infection. On the whole, this workprovides a key base for structural and functional study of SdrE in future,which is necessary for developing new antibacterial drugs.