Béhar Ghislaine, Pacheco Sabino, Maillasson Mike, Mouratou Barbara, Pecorari Frédéric
J Biotechnol. 2014 Dec 20;192 Pt A:123-9. doi: 10.1016/j.jbiotec.2014.10.006.
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.
作为生物技术应用中的一种有用试剂，支架蛋白需要尽可能稳定以确保更长的使用寿命。我们已开发出“7 kDa DNA结合”家族的古菌嗜极端微生物蛋白作为支架，以衍生亲和蛋白（Affitins）。在本研究中，我们评估了一种合理的结构/序列引导方法，通过将其人类IgG结合位点转移到热稳定性更高的Sso7d同源物框架上，来稳定源自Sac7d的Affitin。获得的嵌合体具有功能，在大肠杆菌中表达良好，但热稳定性低于原始Affitin（T(m) = 74.2 °C对T(m) = 80.4 °C）。将两个描述为热稳定野生型Sso7d的单突变引入嵌合体。只有双突变几乎恢复了热稳定性（T(m) = 76.9 °C）。有趣的是，嵌合体及其双突变体在pH 0至至少pH 13范围内都是稳定的。我们的结果表明，在保留其有利特性的同时，有可能进一步提高Affitins对碱性条件（+2个pH单位）的稳定性。由于Affitins基于来自古菌嗜极端微生物的同源物不断增加的家族，我们得出结论，这种方法为它们的改进提供了新的潜力，这将在要求苛刻的生物技术应用中有用。
