An intrinsically stable antibody scFv fragment can tolerate the loss of both disulfide bonds and fold correctly.
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.
Wörn
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Biochemisches Institut, Universität Zürich, Switzerland.
Plückthun
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D019943:Amino Acid Substitution; D015374:Biosensing Techniques; D002850:Chromatography, Gel; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D017354:Point Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
D004220:Disulfides; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D014508:Urea; D003545:Cysteine