Effect of the order of antibody variable regions on the expression of the single-chain HyHEL10 Fv fragment in E. coli and the thermodynamic analysis of its antigen-binding properties.
In order to physically stabilize the Fv fragment of anti-lysozyme monoclonal antibody, HyHEL10, the variable domains were linked covalently with a flexible linker. A marked difference in the level of expression in E. coli was observed between VH-linker-VL (scFvHL) and VL-linker-VH (scFvLH). The highly expressed scFvLH was purified by a single step of affinity chromatography from the culture supernatant with a typical yield of 3-5 mg per liter of culture. This HyHEL10 scFvLH showed reduced binding activity toward its antigen, HEL, in comparison with Fv. Thermodynamic study showed that this reduced activity was due to entropic loss upon binding to its antigen, although this interaction between scFvLH and its antigen was enthalpically favorable.
Tsumoto
K
K
Department of Chemistry and Biotechnology, Faculty of Engineering, University of Tokyo, Japan.
Nakaoki
Y
Y
Ueda
Y
Y
Ogasahara
K
K
Yutani
K
K
Watanabe
K
K
Kumagai
I
I
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D000937:Antigen-Antibody Reactions; D015153:Blotting, Western; D002151:Calorimetry; D002645:Chickens; D003001:Cloning, Molecular / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D007700:Kinetics; D008969:Molecular Sequence Data; D009113:Muramidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology*; D016254:Mutagenesis, Insertional; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D009113:Muramidase