Abstract
A fully functional cysteine-free derivative of the intrinsically stable anti-HER2 scFv fragment hu4D5-8 was generated by replacing the disulfide forming cysteine residues in VH and VL with the amino acid combination valine-alanine in both domains. The antigen binding properties, determined by ELISA and BIAcore measurements, were not affected by removal of the disulfide bonds. The thermodynamic stability of the disulfide-containing scFv of 8.1 kcal/mol is decreased upon complete reduction of both disulfides to 2.7 kcal/mol, while that of the valine-alanine variant is somewhat higher (about 3.8 kcal/ mol). Our results suggest that, in principle, a disulfide-free fully functional derivative of any scFv can be obtained, as long as the corresponding disulfide-containing scFv has a high enough thermodynamic stability.
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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Biosensing Techniques
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Chromatography, Gel
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Cysteine / chemistry
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Disulfides / chemistry*
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Enzyme-Linked Immunosorbent Assay
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Immunoglobulin Fragments / biosynthesis
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Immunoglobulin Fragments / chemistry*
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Immunoglobulin Fragments / isolation & purification
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Immunoglobulin Heavy Chains / biosynthesis
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Immunoglobulin Heavy Chains / chemistry
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Immunoglobulin Heavy Chains / isolation & purification
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Immunoglobulin Light Chains / biosynthesis
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Immunoglobulin Light Chains / chemistry
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Immunoglobulin Light Chains / isolation & purification
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Immunoglobulin Variable Region / biosynthesis
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Immunoglobulin Variable Region / chemistry*
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Immunoglobulin Variable Region / isolation & purification
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Point Mutation
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Protein Denaturation
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Protein Folding*
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / chemistry
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Recombinant Proteins / isolation & purification
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Thermodynamics
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Urea / pharmacology
Substances
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Disulfides
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Immunoglobulin Fragments
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Immunoglobulin Heavy Chains
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Immunoglobulin Light Chains
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Immunoglobulin Variable Region
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Recombinant Proteins
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immunoglobulin Fv
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Urea
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Cysteine