Correct disulfide pairing and efficient refolding of detergent-solubilized single-chain Fv proteins from bacterial inclusion bodies.
In vitro folding of denatured proteins has remained an inefficient and empirical process that has limited the use of bacterially expressed recombinant proteins. In this paper we show that in vitro folding of recombinant single-chain Fv (sFv) proteins is markedly facilitated when disulfide bonds are formed in detergent solution. sFv proteins from three different antibodies were expressed as bacterial cytoplasmic inclusion bodies and solubilized in the weak ionic detergent, sodium lauroylsarcosine (SLS). Upon oxidation in air in the presence of metal ion catalysts, all three sFvs quantitatively formed intrachain disulfide bonds which ran as a single band in SDS-polyacrylamide gel electrophoresis under non-reducing conditions. By contrast, oxidation from 6 M urea gave large amounts of disulfide linked aggregates, and three closely spaced bands of monomeric protein. Detergent was removed from the oxidized sFvs by addition of 6 M urea and absorption with an ion exchange resin. After dialysis and gel filtration in non-denaturing solution, moderate to high yields of monomeric sFv were obtained, depending upon the sFv. All three sFvs gave single bands on isoelectric focussing and SDS-PAGE gels and had similar or identical binding specificities and affinities as the parental Fabs, implying that the final products contained correctly paired disulfide bonds. The correct disulfide pairing suggests that the disulfide loops within the detergent-solubilized sFvs adopt a native-like structure.
Kurucz
I
I
Experimental Immunology Branch, National Cancer Institute, Bethesda, MD 20892-1360, USA.
Titus
J A
JA
Jost
C R
CR
Segal
D M
DM
D000818:Animals; D000907:Antibodies, Bacterial / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D003902:Detergents; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics; D002479:Inclusion Bodies / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis
D000907:Antibodies, Bacterial; D003902:Detergents; D004220:Disulfides; D006146:Guanidines; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D019791:Guanidine