Design of pH Sensitive Binding Proteins from the Hyperthermophilic Sso7d Scaffold

Abstract

We have engineered pH sensitive binding proteins for the Fc portion of human immunoglobulin G (hIgG) (hFc) using two different strategies – histidine scanning and random mutagenesis. We obtained an hFc-binding protein, Sso7d-hFc, through mutagenesis of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus; Sso7d-hFc was isolated from a combinatorial library of Sso7d mutants using yeast surface display. Subsequently, we identified a pH sensitive mutant, Sso7d-his-hFc, through systematic evaluation of Sso7d-hFc mutants containing single histidine substitutions. In parallel, we also developed a yeast display screening strategy to isolate a different pH sensitive hFc binder, Sso7d-ev-hFc, from a library of mutants obtained by random mutagenesis of a pool of hFc binders. In contrast to Sso7d-hFc, both Sso7d-his-hFc and Sso7d-ev-hFc have a higher binding affinity for hFc at pH 7.4 than at pH 4.5. The Sso7d-mutant hFc binders can be recombinantly expressed at high yield in E. coli and are monomeric in solution. They bind an epitope in the CH3 domain of hFc that has high sequence homology in all four hIgG isotypes (hIgG1–4), and recognize hIgG1–4 as well as deglycosylated hIgG in western blotting assays. pH sensitive hFc binders are attractive candidates for use in chromatography, to achieve elution of IgG under milder pH conditions. However, the surface density of immobilized hFc binders, as well as the avidity effect arising from the multivalent interaction of dimeric hFc with the capture surface, influences the pH dependence of dissociation from the capture surface. Therefore, further studies are needed to evaluate if the Sso7d mutants identified in this study are indeed useful as affinity ligands in chromatography.

Inefficient Ribosomal Skipping Enables Simultaneous Secretion and Display of Proteins in Saccharomyces cerevisiae

“…The difference in signal between Sso7dstrep and Sso7dhFc is likely due to the difference in binding affinities for their respective targets. The binding affinity (K D ) of Sso7dstrep for streptavidin of 12 nM, as determined in experiments where Sso7dstrep was immobilized 17 ; on the other hand, the K D of Sso7dhFc for hFc as determined in experiments where hFc is immobilized is 400 nM 19 .…”

“…Towards this end, we sought to isolate binders with higher affinity for a model target protein (hen egg lysozyme) from a library generated by random mutagenesis of a pool of low affinity binders. DNA from a pool of low affinity binders to lysozyme obtained by magnet activated cell sorting (MACS) of an Sso7d library of ~ 10 8 mutants 17 was used to generate a yeast display library as previously described 19 , using the pCT-NT-F2A vector; the library diversity was estimated as ~ 5×10 7 mutants.…”

“…Sso7d sequences from the isolated DNA were amplified by error-prone PCR using nucleotide analogs, with primers Pf2 and Pr2, as detailed by Gera et al 19 . Products of the error-prone PCR were combined, and further amplified by PCR using primers Pf4 and Pr4 in six identical 50 μL reactions.…”

Derivative of Extremophilic 50S Ribosomal Protein L35Ae as an Alternative Protein Scaffold

“…Overall, unlike the original L35Ae 10X protein, both L4 and L7 possess submicromolar to micromolar affinity to HEL, and cross-react with its homologue, despite multimerization at high concentrations (Table 2). Cross-reactivity with related proteins was reported for some of novel binding proteins, including those based on DARPin and Sso7d frameworks [26, 52]. …”

Activity-based assessment of an engineered hyperthermophilic protein as a capture agent in paper-based diagnostic tests

“…Because the Sso7d scaffold is immuno-orthogonal, there is limited risk of cross-reaction with immunological factors found in patient samples. Sso7d has also demonstrated activity following surface immobilization, 37 and features a small molecular footprint (r g = 1.31 nm), potentially allowing for higher molecular surface densities and enhanced substrate functionalization. Thus, the Sso7d scaffold meets many of the criteria required of a binding protein applied in point-of-care diagnostic biosensors (Figure 1).…”