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 Šebo^1,2

petr.maly@img.cas.cz

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 K_d 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.