Engineering antibody fragments to fold in the absence of disulfide bonds.
Disulfide bonds play a critical role in the stabilization of the immunoglobulin beta-sandwich sandwich. Under reducing conditions, such as those that prevail in the cytoplasm, disulfide bonds do not normally form and as a result most antibodies expressed in that compartment (intrabodies) accumulate in a misfolded and inactive state. We have developed a simple method for the quantitative isolation of antibody fragments that retain full activity under reducing conditions from large mutant libraries. In E. coli, inactivation of the cysteine oxidoreductase DsbA abolishes protein oxidation in the periplasm, which leads to the accumulation of scFvs and other disulfide-containing proteins in a reduced form. Libraries of mutant scFvs were tethered onto the inner membrane of dsbA cells and mutants that could bind fluorescently labeled antigen in the reducing periplasm were screened by Anchored Periplasmic Expression (APEx; Harvey et al., Proc Natl Acad Sci USA 2004;101:9193-9198.). Using this approach, we isolated scFv antibody variants that are fully active when expressed in the cytoplasm or when the four Cys residues that normally form disulfides are substituted by Ser residues.
Seo
Min Jeong
MJ
Department of Chemical Engineering, University of Texas, Austin, 78712, USA.
Jeong
Ki Jun
KJ
Leysath
Clinton E
CE
Ellington
Andrew D
AD
Iverson
Brent L
BL
Georgiou
George
G
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000276:immunology / Q000378:metabolism*; D001427:Bacterial Toxins / Q000276:immunology / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D019020:Directed Molecular Evolution; D004220:Disulfides / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics; D005434:Flow Cytometry; D015723:Gene Library; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases / Q000235:genetics; D015202:Protein Engineering; D017510:Protein Folding; D016415:Sequence Alignment; D012694:Serine / Q000378:metabolism
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D004220:Disulfides; D029968:Escherichia coli Proteins; D007135:Immunoglobulin Variable Region; C030325:anthrax toxin; D012694:Serine; D019704:Protein Disulfide-Isomerases; C526648:dsbA protein, E coli; D003545:Cysteine