Switching an anti-IgG binding site between archaeal extremophilic proteins results in Affitins with enhanced pH stability

Abstract

As a useful reagent for biotechnological applications, a scaffold protein needs to be as stable as possible to ensure longer lifetimes. We have developed archaeal extremophilic proteins from the “7 kDa DNA-binding” family as scaffolds to derive affinity proteins (Affitins). In this study, we evaluated a rational structure/sequence-guided approach to stabilize an Affitin derived from Sac7d by transferring its human IgG binding site onto the framework of the more thermally stable Sso7d homolog. The chimera obtained was functional, well expressed in Escherichia coli, but less thermally stable than the original Affitin (T(m) = 74.2 °C vs. T(m) = 80.4 °C). Two single mutations described as thermally stabilizing wild type Sso7d were introduced into chimeras. Only the double mutation nearly restored thermal stability (T(m) = 76.9 °C). Interestingly, the chimera and its double mutant were stable from pH 0 up to at least pH 13. Our results show that it is possible to increase further the stability of Affitins toward alkaline conditions (+2 pH units) while conserving their advantageous properties. As Affitins are based on a growing family of homologs from archaeal extremophiles, we conclude that this approach offers new potential for their improvement, which will be useful in demanding biotechnological applications.

The archaeal “7 kDa DNA-binding” proteins: extended characterization of an old gifted family

“…We previously reported that Sac7d and Sso7d are highly resistant from low to high pH1213, and here, we confirm this as a general property of the “7 kDa DNA-binding” proteins. This is interesting considering that the pH inside Sulfolobus cells is about 6.55 while the pH outside is 3.533, suggesting there has been no selection pressure for these pH resistances.…”

“…Also, a smaller size is interesting for in vivo applications, such as imaging and therapy, as it allows a better diffusion across barriers or to reach tumors for example44. Up to now, Sac7d and Sso7d have been used as scaffolds to design Affitins13212425. However, this study teaches us that Sto7 and Aho7c should be a good basis to generate more robust Affitins.…”

“…Circular dichroism (CD) spectra and the monitoring of ellipticity vs pH were done for each protein as published elsewhere for Affitins1213.…”

“…They have been characterized for their structure, function, chemical stability and biophysical properties7. Sac7d and Sso7d are hyperthermostable ( T m  = 90.4 °C and 100.2 °C, respectively)1011 and are resistant from pH 0 up to at least pH 121213. Although Sac7d and Sso7d sequences show only few differences, Sso7d is more stable than Sac7d.…”

A novel, smaller scaffold for Affitins: Showcase with binders specific for EpCAM

“…We have previously described Affitins as artificial affinity proteins derived from the archaeal extremophilic 7 kDa DNA‐binding proteins, such as Sac7d and Sso7d (Béhar et al, ; Krehenbrink et al, ; Mouratou et al, ; Pecorari & Alzari, ). Recently, we have also extensively characterized this family of proteins (Kalichuk et al, ).…”