HSP90 C Domain Structure And Function Study Of C.albicans

C.albicans is a symbiotic fungus in human,it can turn to the pathogenic fugus state,the clinic survey show that C.albicans has became the most prevail pathogenic fugus in world.The C.albicans infects people mainly by invaded internal orgens and blood,the drug resistance and the virulence traits of C.albicans become more and more stronger for these year.Hsp90 participates the pck1,this is a meaningful machine to enhance the fugal grow velocity and the drug resisitance of azoles.we found tha the CaHsp90 has 65% homology to hHsp90 by computer analysis software,but the C domain has huge discrepancy between CaHsp90 and hHsp90,the discrepancy offers the targets to decline the drug resistance of candidas especially.Based on the sequence discrepancy,we found the CaHsp90(1-673)and CaHsp90(1-655)are key sequence to Hsp90 dimeric structure through software analysis result and documents.CaHsp90(1-673)and CaHsp(1-655)correspond to hHsp90(1-696)and Hsp90(1-679),then we research the efficient antifugal drug targets based on dicrepancy of CaHsp90 and hHsp90.We constructed the plamids,and put the CaHsp90 sequence CaHsp90(1-673)sequence and CaHsp90(1-655)sequences of wild type Sc5314 strain into PET22 b by enzyme digestions technique successfull.CaHSP90,CaHSP90(1-673),CaHSP90(1-655),hHSP90,hHSP90(1-679)and hHSP90(1-696)were expressed in E.coli BL21(DE3)cells and the His-tagged proteins were purified through Ni2+-NTA column.Finally CaHSP90,CaHSP90(1-673),CaHSP90(1-655),hHSP90,hHSP90(1-679)and hHSP90(1-696)through Superdex-200 column chromatography on FPLC,the molecular weights and oligomeric forms of these proteins were analyzed by SDS-PAGE and size exclusion chromatography(SEC).Rsult:we can construct the plamids successful by PCR technique and company sequencing,we found the plamids can duplicate and express in E.coli BL21 by SDS-PAGE and size exclusion chromatography(SEC).we found the discrepancy between CaHsp(1-655)and hHsp(1-679)through Superdex-200 column chromatography on FPLC,CaHsp90(1-655)ranges monomer from dimer,and hHsp90 belongs to dimer.CaHSP90,CaHSP90(1-673),hHSP9 and hHSP90(1-696)belong to oligomer.High temperature and the change of C domain structure contribute to oligomer,so the inter machine should be studied,we also found the protein structure changing of CaHSP90 influlence on chemical modification the protein,finally,we have the ATPase activity test。...