title: Simultaneous removal of T and B cell epitope in recombinant staphylokinase by structure-based mutagenesis of immuno-dominant Arg77 and Glu80 residues.
abstract: OBJECTIVE
  To reduce immunogenicity of recombined staphylokinase (r-Sak) , site-directed mutagenesis of Arg77 and Glu80 residue was performed to simultaneously remove T and B cell epitope in r-Sak molecule.
  
  
  METHODS
  The solvent accessible surface areas of residues 77 and 80 in r-Sak were used to analyze rational design of Sak mutation. The Sak mutants were expressed in E coli DH5alpha. After purified by a 3-step chromatography, their fibrinolytic activities and immunological properties were analyzed.
  
  
  RESULTS
  Immunogenicity tests suggested that Sak induced a Th2-type immune response. Substitution of Glu80 with alanine or serine successfully reduced its solvent accessible surface area while simultaneously removing part of the T and B cell epitope. Changing Arg77 to glutamine, asparagine, or lysine removed only part of the T cell epitope. Of six dually substituted variants, Sak (R77Q/E80A) and Sak(R77Q/E80S) variants effectively eliminated part of the B and T cell epitopes, which markedly reduced their immunogenicity.
authors.affiliation: College of Life Science, Hebei Normal University, Shijiazhuang 050016, China. xuruiguang_1984@163.com
authors.forename: Ruiguang
authors.initials: R
authors.lastname: Xu
authors.forename: Jintian
authors.initials: J
authors.lastname: He
authors.forename: Kai
authors.initials: K
authors.lastname: Jia
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.forename: Jianwei
authors.initials: J
authors.lastname: Liu
authors.forename: Ke
authors.initials: K
authors.lastname: Zhu
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D001666:Binding Sites, Antibody; D049109:Cell Proliferation / Q000187:drug effects; D018985:Epitopes, B-Lymphocyte / Q000235:genetics* / Q000276:immunology; D018984:Epitopes, T-Lymphocyte / Q000235:genetics* / Q000276:immunology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000276:immunology* / Q000302:isolation & purification / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D015847:Interleukin-4 / Q000378:metabolism; D008666:Metalloendopeptidases / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology
chemical_list: D000907:Antibodies, Bacterial; D018985:Epitopes, B-Lymphocyte; D018984:Epitopes, T-Lymphocyte; D005343:Fibrinolytic Agents; D011994:Recombinant Proteins; D015847:Interleukin-4; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus