Pacheco Sabino, Béhar Ghislaine, Maillasson Mike, Mouratou Barbara, Pecorari Frédéric
CRCNA - UMR 892 INSERM, 8 quai Moncousu, BP 70721, 44007 Nantes, Cedex 1, France 6299 CNRS, 8 quai Moncousu, BP 70721, 44007 Nantes, Cedex 1, France University of Nantes, 8 quai Moncousu, BP 70721, 44007 Nantes, Cedex 1, France Institut Pasteur, CNRS UMR 3528, Unité de Microbiologie Structurale, 25 rue du Dr. Roux, 72724 Paris Cedex 15, France.
CRCNA - UMR 892 INSERM, 8 quai Moncousu, BP 70721, 44007 Nantes, Cedex 1, France 6299 CNRS, 8 quai Moncousu, BP 70721, 44007 Nantes, Cedex 1, France University of Nantes, 8 quai Moncousu, BP 70721, 44007 Nantes, Cedex 1, France.
Protein Eng Des Sel. 2014 Oct;27(10):431-8. doi: 10.1093/protein/gzu042.
Artificially transforming a scaffold protein into binders often consists of introducing diversity into its natural binding region by directed mutagenesis. We have previously developed the archaeal extremophilic Sac7d protein as a scaffold to derive affinity reagents (Affitins) by randomization of only a flat surface, or a flat surface and two short loops with natural lengths. Short loops are believed to contribute to stability of extremophilic proteins, and loop extension has been reported detrimental for the thermal and chemical stabilities of mesophilic proteins. In this work, we wanted to evaluate the possibility of designing target-binding proteins based on Sac7d by using a complementary determining region (CDR). To this aim, we inserted into three different loops a 10 residues CDR from the cAb-Lys3 anti-lysozyme camel antibody. The chimeras obtained were as stable as wild-type (WT) Sac7d at extreme pH and their structural integrity was supported. Chimeras were thermally stable, but with T(m)s from 60.9 to 66.3°C (cf. 91°C for Sac7d) which shows that loop extension is detrimental for thermal stability of Sac7d. The loop 3 enabled anti-lysozyme activity. These results pave the way for the use of CDR(s) from antibodies and/or extended randomized loop(s) to increase the potential of binding of Affitins.
将支架蛋白人工转化为结合蛋白通常包括通过定向诱变在其天然结合区域引入多样性。我们之前已开发出嗜热古菌的Sac7d蛋白作为支架，仅通过一个平面或一个平面以及两个天然长度的短环的随机化来衍生亲和试剂（Affitins）。短环被认为有助于嗜热蛋白的稳定性，并且据报道环的延伸对中温蛋白的热稳定性和化学稳定性不利。在这项工作中，我们想评估利用互补决定区（CDR）基于Sac7d设计靶标结合蛋白的可能性。为此，我们将来自骆驼cAb-Lys3抗溶菌酶抗体的10个残基的CDR插入到三个不同的环中。获得的嵌合体在极端pH条件下与野生型（WT）Sac7d一样稳定，并且其结构完整性得到了支持。嵌合体具有热稳定性，但其熔解温度（Tm）为60.9至66.3°C（相比之下，Sac7d为91°C），这表明环的延伸对Sac7d的热稳定性不利。环3具有抗溶菌酶活性。这些结果为使用来自抗体的CDR和/或延伸的随机环来提高Affitins的结合潜力铺平了道路。
