Biochemisches Institut, Universität Zürich, Zürich, Switzerland.
Protein Eng Des Sel. 2010 Apr;23(4):175-84. doi: 10.1093/protein/gzp087. Epub 2010 Feb 3.
Directed evolution is a powerful approach for isolating high-affinity binders from complex libraries. In affinity maturation experiments, binders with the highest affinities in the library are typically isolated through selections for decreased off rate using a suitable selection platform (e.g. phage display or ribosome display). In such experiments, the library is initially exposed to biotinylated antigen and the binding reaction is allowed to proceed. A large excess of unbiotinylated antigen is then added as a competitor to capture the vast majority of rapidly dissociating molecules; the slowly dissociating library members can subsequently be rescued by capturing the biotin-carrying complexes. To optimize the parameters for such affinity maturation experiments, we performed both deterministic and stochastic simulations of off-rate selection experiments using different input libraries. Our results suggest that the most critical parameters for achieving the lowest off rates after selection are the ratio of competitor antigen to selectable antigen and the selection time. Furthermore, the selection time has an optimum that depends on the experimental setup and the nature of the library. Notably, if selections are carried out for times much longer than the optimum, equilibrium is reached and the selection pressure is weakened or lost. Comparison of different selection strategies revealed that sequential selection rounds with lower stringency are favored over high-stringency selection experiments due to enhanced diversity in the selected pools. Such simulations may be helpful in optimizing affinity maturation strategies and off-rate selection experiments.
定向进化是从复杂文库中分离高亲和力结合物的一种强大方法。在亲和力成熟实验中，通常通过选择降低离解速率来从文库中分离具有最高亲和力的结合物，使用合适的选择平台（例如噬菌体展示或核糖体展示）。在这样的实验中，文库最初暴露于生物素化抗原，允许结合反应进行。然后加入大量未生物素化的抗原作为竞争物，以捕获大多数快速解离的分子；随后可以通过捕获携带生物素的复合物来挽救缓慢解离的文库成员。为了优化此类亲和力成熟实验的参数，我们使用不同的输入文库对离解速率选择实验进行了确定性和随机模拟。我们的结果表明，在选择后达到最低离解速率的最关键参数是竞争抗原与可选择抗原的比例和选择时间。此外，选择时间具有一个最佳值，该最佳值取决于实验设置和文库的性质。值得注意的是，如果选择时间远远超过最佳值，就会达到平衡，选择压力会减弱或丧失。不同选择策略的比较表明，由于所选池中的多样性增强，低严格性的连续选择轮次优于高严格性的选择实验。这种模拟可能有助于优化亲和力成熟策略和离解速率选择实验。
