The final His6-REX-TolA-AVI fusion proteins were produced as biotinylated proteins in the BL21 (DE3) BirA strain expressing biotin ligase (BirA) in the presence of 50 d-biotin in the LB medium and following induction with 2 mIPTG. for IL-23R binding in ELISA were confirmed to recognize human being IL-23R-IgG in surface plasmon resonance experiments, estimating the binding affinity in the sub- to nanomolar range. We further shown that several REX variants bind to human being leukemic cell lines K-562, THP-1 and Jurkat, and this binding correlated NVP-BSK805 with IL-23R cell-surface manifestation. The REX125, REX009 and NVP-BSK805 REX128 variants competed with the p19 protein for binding to THP-1 cells. Moreover, the presence of REX125, REX009 and REX115 variants significantly inhibited the IL-23-driven growth of IL-17-generating main human being CD4+ T-cells. Therefore, we conclude that unique IL-23R antagonists derived from the ABD scaffold were generated that might be useful in developing novel anti-inflammatory biologicals. Proteins 2014; 82:975C989. detection, diagnostics or high-affinity bioanalytical methods.27C29 Preservation of folding function together with easy scaffold modifications and low molecular weight, allowing excellent tissue penetration, move the Affibody-derived binders close to the therapeutic use. The albumin-binding website of streptococcal protein G30C33 is definitely another example of three-helix package scaffold being successfully utilized for the building of combinatorial libraries. Recently we have shown that randomization of 11 residues of a flat helical surface, created by two helices with an inter-link loop (Fig. ?(Fig.2),2), was sufficient to yield a combinatorial library of a theoretical difficulty of 1016 codon variants that was then successfully utilized for the selection of high-affinity binders of human being IFN-.34 In this type of library, organic HSA-binding affinity of the ABD website was compromised in favor of newly engineered affinity for the chosen target. On the other hand, another group randomized 11 residues of a different ABD scaffold surface to generate a combinatorial library that yielded fresh affinity yet maintained the original HSA binding. This type of dual-affinity library was used to select binders of human being TNF-35 and ErbB3.36 Open in a separate window Number 2 Location of randomized positions in the ABD scaffold. The protein structure of the ABD CMKBR7 website of streptrococcal protein G (PDBID 1GJT) is definitely demonstrated in ribbon representation, with the C positions of the 11 residues selected for randomization demonstrated as yellow spheres. IL-23 receptor belongs to the class-I cytokine receptor family and shares standard features with tandem fibronectin-type III (FnIII) domains comprising a hallmark pattern NVP-BSK805 of disulfide bonds and WQPWS sequence tag much like a conserved WSXWS cytokine receptor consensus located in the transmembrane-proximal FnIII website.10 Both domains form a cytokine-binding homology region (CHR) which, in concert with a terminal Ig-like domain, is believed to play a substantial role in IL-23 binding. The molecular structure of the IL-23/IL-23R complex is not available yet, therefore, developing efficient inhibitors of IL-23 function having a encouraging therapeutic potential remains cumbersome. Here we describe generation of a set of novel recombinant antagonists of the human being IL-23 receptor. Their inhibitory potency on IL-23 function is definitely demonstrated on several plans of binding assays, cell-surface competition experiments and practical assays. Our data further document the three-helix package scaffold of ABD is suitable for development of anti-inflammatory IL-23 receptor-based next generation therapeutics. MATERIALS AND METHODS Antibodies and detection providers Monoclonal antibodies (mAbs) anti-human IL-23R-allophycocyanin (APC) (mouse IgG2b) specific for the human being IL-23 receptor and IgG2b isotype control-APC (mouse IgG2b) were from R&D Systems, Minneapolis, MN. Mouse anti-p19 mAb was purchased from Biolegend, San Diego, CA. Cy5-conjugated goat anti-mouse IgG (F(ab)2 fragment) was from Jackson ImmunoResearch Laboratories, Western Grove, PA. Streptavidin-phycoerythrin was purchased from eBioscience, San Diego, CA. Cell lines and growth conditions The cell lines used in the experiments were a human acute monocytic leukemia cell line, THP-1 NVP-BSK805 (ATCC number: TIB-202), a human leukemic cell.