Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library.
Structure-guided phage display was used to select for combinations of interface residues for antibody C(H)3 domains that promote the formation of stable heterodimers. A C(H)3 "knob" mutant was made by replacement of a small residue, threonine, with a larger one, tryptophan: T366W. A library of C(H)3 "hole" mutants was then created by randomizing residues 366, 368 and 407, which are in proximity to the knob on the partner C(H)3 domain. The C(H)3 knob mutant was fused to a peptide flag and the C(H)3 hole library was fused to M13 gene III. Phage displaying stable C(H)3 heterodimers were recovered by panning using an anti-flag antibody. Phage-selected C(H)3 heterodimers differed in sequence from the previously designed heterodimer T366W-Y407'A, and most clones tested were more stable to guanidine hydrochloride denaturation. The thermal stability of individual C(H)3 domains secreted from Escherichia coli was analyzed by differential scanning calorimetry. One heterodimer, T366W-T366'S:L368'A:Y407'V, had a t(m) of 69.4 degrees C, which is 4.0 deg.C higher than that for the designed heterodimer and 11.0 deg.C lower than that for the wild-type homodimer. The phage-selected C(H)3 mutant maintained the preference for forming heterodimers over homodimers as judged by near-quantitative formation of an antibody/immunoadhesin hybrid in a cotransfection assay. Phage optimization provides a complementary and more comprehensive strategy to rational design for engineering homodimers for heterodimerization.
Atwell
S
S
Department of Protein Engineering, Genentech Inc., South San Francisco, CA 94080, USA.
Ridgway
J B
JB
Wells
J A
JA
Carter
P
P
D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017104:Bacteriophage M13 / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D017252:CD3 Complex / Q000235:genetics / Q000276:immunology; D016860:CD4 Immunoadhesins / Q000235:genetics; D002152:Calorimetry, Differential Scanning; D019281:Dimerization; D015723:Gene Library; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry / Q000494:pharmacology; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D014162:Transfection
D000906:Antibodies; D017252:CD3 Complex; D016860:CD4 Immunoadhesins; C096753:CD4-IgG(2); D006146:Guanidines; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D011994:Recombinant Proteins; D019791:Guanidine