Monovalent phage display of human interleukin (hIL)-6: selection of superbinder variants from a complex molecular repertoire in the hIL-6 D-helix.
Phage display of proteins can be used to study ligand-receptor interaction and for the affinity-maturation of binding sites in polypeptide hormones and/or cytokines. We have expressed human interleukin-6 (hIL-6) on M13 phage in a monovalent fashion as a fusion protein with the phage coat protein, pIII. Phage-displayed hIL-6 is correctly folded, as judged by its ability to interact with conformation-specific anti-hIL-6 monoclonal antibodies (mAb) and with the hIL-6 receptor complex in vitro. We set up an experimental protocol for the efficient affinity selection of hIL-6 phage using the extracellular portion of the hIL-6 receptor alpha (hIL-6R alpha) fixed on a solid phase. This system was used to affinity-purify from a library of hIL-6 variants, in which four residues in the predicted D-helix of the cytokine were fully randomized, mutants binding hIL-6R alpha with higher efficiency than the wild type. When the best-binder variant Q175I/Q183A was combined with a previously identified superbinder S176R [Savino et al., Proc. Natl. Acad. Sci. 90 (1993) 4067-4071], a triple-substitution mutant Q175I/S176R/Q183A (hIL-6IRA) was obtained with a fivefold increased hIL-6R alpha binding and a 2.5-fold enhanced biological activity.
Cabibbo
A
A
Department of Genetics, Istituto di Ricerche di Biologia Moleculare P. Angeletti (IRBM), Pomezìa (Roma), Italy.
Sporeno
E
E
Toniatti
C
C
Altamura
S
S
Savino
R
R
Paonessa
G
G
Ciliberto
G
G
D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000378:metabolism*; D017104:Bacteriophage M13; D050822:Cytokine Receptor gp130; D006801:Humans; D015850:Interleukin-6 / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018121:Receptors, Cytokine / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; C494199:IL6ST protein, human; D015850:Interleukin-6; D008562:Membrane Glycoproteins; D018121:Receptors, Cytokine; D011994:Recombinant Proteins; D050822:Cytokine Receptor gp130