| Specific and non-covalent interaction between species, referred to as molecular recognition, is central to several diverse biological processes. Antibodies have been extensively used as binding molecules for molecular recognition. However, their applications are limited by low thermodynamic stability, large size and expensive production. Therefore, there is a keen interest in alternative non-immunoglobulin scaffold frameworks. Hyperthermophilic organisms are a rich source of small proteins with no cysteines that are excellent candidates for molecular recognition scaffolds.;We have shown that highly stable binding proteins for a wide spectrum of targets can be generated through mutagenesis of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus. Sso7d is a small (∼ 7 kDa, 63 amino acids) DNA binding protein that lacks cysteine residues and has a melting temperature of nearly 100°C. Sso7d-derived mutants have high thermal stability, resistant to chemical denaturation by guanidine hydrochloride and retain their secondary structure after extended incubation in extreme pH conditions.;The high thermal, chemical and pH stability of Sso7d-derived proteins is particularly useful for chromatographic separations. Therefore, to further explore the use of Sso7d-based ligands in the context of protein purification, we isolated Sso7d-based protein ligands that bind the Fc portion of human IgG (hFc). These hFc binders could be used to isolate human IgG from complex media. We systematically characterized the binding of the Sso7d-based ligands to various hIgG isotypes as well the effect of glycosylation on binding. Further, using a combination of histidine scanning mutagenesis and directed evolution, we isolated Sso7d variants that can elute human IgG under milder conditions.;Lastly, to further explore the potential of hyperthermophilic proteins as scaffolds, we generated a library-of-libraries, or a combinatorial "super-library", of ∼ 4 x 108 proteins by randomizing surface accessible residues on seven different non-immunoglobulin scaffolds of hyperthermophilic origin. Binding proteins with high specificity and to a diverse set of model targets could be isolated from this super-library. Interestingly, the pool of highest affinity binders for each target contained proteins derived from a distinct subset of scaffolds, suggesting that certain scaffolds may be more suited to generate binders to a specific target. Also strikingly, binders from the super library have higher or similar affinities than those from a library with three orders of magnitude greater overall diversity, but derived by randomizing the previously validated Sso7d scaffold. Therefore, this approach produces a combinatorial library of higher quality. Our results are particularly relevant since current screening technologies sample only a fraction of the theoretical diversity generated by randomizing 10-15 residues. Further, mutant proteins from multiple scaffolds described in this study have favorable properties such as low molecular weight ( 74 °C) and ease of recombinant expression in E. coli. . |
