Highly efficient recovery of functional single-chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled introduction of oxidizing reagent--application to a human single-chain Fv fragment.
An improved and efficient refolding system for a single-chain antibody fragment (scFv) from inclusion bodies expressed in Escherichia coli was developed. Stepwise removal of denaturing reagent and controlled addition of oxidizing reagent were found to be the most effective conditions to achieve for almost complete recovery of functional monomeric scFv from inclusion bodies. Adding L-arginine to the refolding solution also increased the yield of refolded functional scFv. The single-chain Fv fragments of both a mouse anti-lysozyme monoclonal antibody, HyHEL10, and a human monoclonal antibody against the D antigen of the Rh blood group, D10, in solubilized inclusion bodies could be refolded under these conditions with yields of up to 95%. The refolding procedures developed in this study will contribute to providing a stable supply of large amounts of human single-chain Fv fragments.
Tsumoto
K
K
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Japan.
Shinoki
K
K
Kondo
H
H
Uchikawa
M
M
Juji
T
T
Kumagai
I
I
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D004220:Disulfides; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D005978:Glutathione / Q000494:pharmacology*; D019791:Guanidine; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D002479:Inclusion Bodies / Q000737:chemistry*; D051379:Mice; D009113:Muramidase / Q000276:immunology / Q000378:metabolism; D016877:Oxidants; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D019163:Reducing Agents; D012204:Rh-Hr Blood-Group System / Q000276:immunology; D012995:Solubility
D000911:Antibodies, Monoclonal; D004220:Disulfides; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D016877:Oxidants; D011994:Recombinant Proteins; D019163:Reducing Agents; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen; C055966:immunoglobulin Fv; D001120:Arginine; D009113:Muramidase; D005978:Glutathione; D019791:Guanidine