Janssen Research & Development, L.L.C., Radnor, PA 19087, USA.
Protein Eng Des Sel. 2012 Mar;25(3):107-17. doi: 10.1093/protein/gzr064. Epub 2012 Jan 12.
The use of consensus design to produce stable proteins has been applied to numerous structures and classes of proteins. Here, we describe the engineering of novel FN3 domains from two different proteins, namely human fibronectin and human tenascin-C, as potential alternative scaffold biotherapeutics. The resulting FN3 domains were found to be robustly expressed in Escherichia coli, soluble and highly stable, with melting temperatures of 89 and 78°C, respectively. X-ray crystallography was used to confirm that the consensus approach led to a structure consistent with the FN3 design despite having only low-sequence identity to natural FN3 domains. The ability of the Tenascin consensus domain to withstand mutations in the loop regions connecting the β-strands was investigated using alanine scanning mutagenesis demonstrating the potential for randomization in these regions. Finally, rational design was used to produce point mutations that significantly increase the stability of one of the consensus domains. Together our data suggest that consensus FN3 domains have potential utility as alternative scaffold therapeutics.
利用共识设计来生产稳定的蛋白质已经被应用于许多结构和蛋白质类别。在这里，我们描述了两种不同蛋白质（即人纤连蛋白和人腱生蛋白 C）的新型 FN3 结构域的工程设计，作为潜在的替代支架生物治疗药物。结果发现，所得到的 FN3 结构域在大肠杆菌中得到了稳健的表达，具有可溶性和高度稳定性，其熔点分别为 89°C 和 78°C。X 射线晶体学用于证实，尽管与天然 FN3 结构域的序列同一性较低，但共识方法导致的结构与 FN3 设计一致。使用丙氨酸扫描诱变研究了 Tenascin 共识结构域在连接 β-链的环区中耐受突变的能力，表明这些区域具有随机化的潜力。最后，通过合理设计产生了点突变，显著提高了一个共识结构域的稳定性。总之，我们的数据表明共识 FN3 结构域具有作为替代支架治疗药物的潜在用途。
