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

Ghislaine Béhar, Sabino Pacheco, Mike Maillasson, Barbara Mouratou, Frédéric Pecorari
Journal of Biotechnology 2014 December 20, 192 Pt A: 123-9
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.


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