title: Refolding of a staphylokinase variant y1-Sak by reverse dilution.
abstract: To develop more potent thrombolytic agents with fibrinolytic and antiplatelet aggregation activity, staphylokinase (Sak) variant Y1-Sak, a recombinant mutant of the Staphylococcus aureus protein Sak, was constructed. Y1-Sak formed an insoluble inclusion body when overexpressed in Escherichia coli strain JF1125. To obtain an optimized refolding process, dilution refolding was used to optimize refolding conditions. The results revealed that additive L: -arginine and refolding temperature played critical roles in the refolding of Y1-Sak. Subsequently, two refolding methods, gel filtration and reverse dilution, were investigated to refold Y1-Sak. The results indicated that the fibrinolytic activity and recovery of Y1-Sak from gel filtration were lower than those from reverse dilution. Reverse dilution refolding successfully reduced the side reaction of refolding with the help of L: -arginine, and the fibrinolytic activity and recovery of Y1-Sak were significantly improved. Functional analysis revealed that refolded Y1-Sak by reverse dilution possessed fibrinolytic and antiplatelet aggregation activities. Moreover, the immunogenicity of Y1-Sak was significantly reduced.
authors.affiliation: College of Life Science, Hebei Normal University, 113 Yuhua East Road, Shijiazhuang City, Hebei Province, 050016, People's Republic of China. he_jintian@yahoo.com
authors.forename: Jintian
authors.initials: J
authors.lastname: He
authors.forename: Gaizhen
authors.initials: G
authors.lastname: Wang
authors.forename: Ruiguang
authors.initials: R
authors.lastname: Xu
authors.forename: Jinlin
authors.initials: J
authors.lastname: Feng
authors.forename: Jinlong
authors.initials: J
authors.lastname: Wang
authors.forename: Huabo
authors.initials: H
authors.lastname: Su
authors.forename: Houyan
authors.initials: H
authors.lastname: Song
mesh_terms: D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005343:Fibrinolytic Agents / Q000737:chemistry*; D014644:Genetic Variation / Q000235:genetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D012996:Solutions; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics
chemical_list: D005343:Fibrinolytic Agents; D011993:Recombinant Fusion Proteins; D012996:Solutions; D008666:Metalloendopeptidases; C525514:staphylokinase SFH