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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Substitution
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Animals
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Antibodies, Bacterial / immunology
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Binding Sites, Antibody
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Cell Proliferation / drug effects
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Epitopes, B-Lymphocyte / genetics*
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Epitopes, B-Lymphocyte / immunology
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Epitopes, T-Lymphocyte / genetics*
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Epitopes, T-Lymphocyte / immunology
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Escherichia coli / enzymology
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Escherichia coli / genetics*
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Escherichia coli / metabolism
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Fibrinolytic Agents / immunology*
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Fibrinolytic Agents / isolation & purification
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Fibrinolytic Agents / pharmacology
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Gene Expression Regulation, Enzymologic
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Interleukin-4 / metabolism
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Metalloendopeptidases / genetics*
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Metalloendopeptidases / immunology
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Metalloendopeptidases / isolation & purification
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Metalloendopeptidases / pharmacology
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Mice
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Mice, Inbred BALB C
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Molecular Structure
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Mutagenesis, Site-Directed / methods
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Recombinant Proteins / genetics*
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Recombinant Proteins / immunology
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / pharmacology
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T-Lymphocytes / drug effects
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T-Lymphocytes / immunology
Substances
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Antibodies, Bacterial
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Epitopes, B-Lymphocyte
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Epitopes, T-Lymphocyte
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Fibrinolytic Agents
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Recombinant Proteins
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Interleukin-4
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Metalloendopeptidases
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auR protein, Staphylococcus aureus