The Research On Expression,Purification And Biochemical Property Of Heat Shock Protein Sse1

Heat shock protein 70kDa,Hsp70,is a classs of highly conserved molecular chaperone which is essential for maintaining cellular protein homeostasis in organisms ranging from bacteria to mammal.Hsp70 plays key role in the refolding of misfolded protein,protein translocation across membranes,preventing protein's aggregation and degradation and assisting the disassembly of protein assemblages.Hsp110,a homolog of Hsp70,is abundant in eukaryotes and has a high level expression in mammal tissues especially in brains.Recently,it becomes clear that Hsp110 act as an essencial nucleotide exchange factor for Hsp70.However,Hsp110 is highly efficient in preventing protein aggragetion but lacks the folding activity shown in Hsp70.Hence,we call Hsp11O "holdase",contrast to Hsp70 the"foldase".Hsp110 is represented in Yeast by the Sse1 and Sse2 proteins.Ssel is expressed at moderately high levels under normal growth and further induced at stress conditions such as heat shock.While Sse2 merely expressed upon stress conditions.Ssel was firstly isolated biologically as a cariculum-binding protein from Yeast and has been crystallized and developed to be a model for the study of Hsp110.In this study,based on the homolog between Hsp110 and Hsp70 and the fully research of Hsp70,firstly,we did the site-directed mutants:Sse1-loopl2,Sse1-loop23,Sse1-loop34,Sse1-loop45,Sse1-loop56,Sse1-loop67,Sse1-loop78,Sse1-loopG466PG472P,to study the exact function sites in Sse1.After sequencing correct and induction in E.coli,we purified all the proteins.Then we did fluorescence anisotropy peptide substrate binding assay to explore their different ability in binding substrate between wild type protein Ssel and all the mutants.Third,we did the growing test in vivo to find the difference in phenotype among the mutants.And then we did the western blot assay to see the expression of Ssel in Yeast.The result shows that Sse1,Sse1-loop 12,Sse1-loop78 has the similar binding affinity.The growing test tells that Sse1-loop 12,Sse1-loop78 shows the same phenotype as wild type Sse1.The two results are consistant except for Ssel-loop45.The western blot indicates that all the proteins expressed normally in Yeast.It is telling the truth that the mutated sites have no effect on the expression of protein.The difference in phenotype may be cused by the lose of proteins function or easily degradation.In a word,we finally have the primary knowledge of Ssel in its function sites.