GENERATION OF ARTIFICIAL BINDERS WITH
AFFINITY TO HUMAN CYTOKINES VIA COMPUTER-ASSISTED MUTAGENESIS OF A STABLE
PROTEIN SCAFFOLD AND RIBOSOME DISPLAY SELECTION
Petr Malý1, Jawid N. Ahmad2, Jingjing Li1, Lada Biedermannová1, Milan Kuchař1, Hana Šípová3, Alena Semerádtová4, Jiří Černý1, Hana Petroková1, Pavel Mikulecký1, Jiří Vondrášek1, Jiří Homola3, Jan Malý4, Radim Osička2, Peter Šebo1,2
1Institute of Biotechnology AS CR, v.v.i. and 2Institute of Microbiology AS CR, v.v.i., Vídeňská 1083, 142 20 Prague, Czech Republic; 3Institute of Photonics and Electronics AS CR, v.v.i., Chaberská 57, 182 51, Prague, Czech Republic; 4Faculty of Science, Jan Evangelista Purkyně University, České Mládeže 8, 400 96 Ústí nad Labem, Czech Republic
Albumine binding domain (ABD), a three-helix bundle of native Streptococcal G protein, represents a promising scaffold for the construction of small artificial ligands with required properties, useful as new diagnostic tools or next generation therapeutics. To generate a set of novel binding proteins with high affinity and specificity to human cytokines, surface residues of the ABD molecule were subjected to a detailed analysis using interaction energy map, calculation of solvent accessibility and in silico alanine scanning, resulting in the identification of key residues for the stability and 11 randomization-accessible amino acid positions. Combinatorial DNA library with a theoretical complexity up to 1014 ABD variants, generated using randomized oligonucleotide primers followed by fusion with cDNA coding for TolA helical helper protein, served as a template for the reverse transcription, followed by in vitro translation and ribosome display selection. After several round of two independent screening campaigns, high affinity ligands for human interferon gamma (hIFNγ) were identified, with Kd ranging from 0.2 to 10 nM. Molecular modeling and docking of four best hIFNγ binders suggested that these ABD variants share a common binding region different from that known for the hIFNγ receptor. The predicted binding model for novel ligands is supported by competition ELISA and SPR using a synthetic form of one of the ABD variants and recombinant form of hIFNγ receptor. Novel ligands represent unique artificial probes for structure-function studies and promising tools for the construction of new biosensors. The ABD library and the established screening approach are currently being used for the generation of novel inhibitory ligands blocking the function of human interleukin-23.
Funding by the grants KAN200520702, GACR P305/10/2184 and GACRP303/10/1849, and the institutional research concept AV0Z50520701.