Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.
Methods. 2013 Mar 15;60(1):3-14. doi: 10.1016/j.ymeth.2012.12.010. Epub 2012 Dec 29.
A set of phage display sorting strategies and validation methodologies are presented that are capable of producing high performance synthetic antibodies (sABs) with customized properties. Exquisite control of antigen and conditions during the phage display selection process can yield sABs that: (1) recognize conformational states, (2) target specific regions of the surface of a protein, (3) induce conformational changes, and (4) capture and stabilize multi-protein complexes. These unique capabilities open myriad opportunities to study complex macromolecular processes inaccessible to traditional affinity reagent technology. We present detailed protocols for de novo isolation of binders, as well as examples of downstream biophysical characterization. The methods described are generalizable and can be adapted to other in vitro direct evolution approaches based on yeast or mRNA display.
本文提出了一套噬菌体展示筛选策略和验证方法，能够产生具有定制特性的高性能合成抗体（sAB）。在噬菌体展示选择过程中对抗原和条件的精细控制可以产生能够：（1）识别构象状态，（2）靶向蛋白质表面的特定区域，（3）诱导构象变化，以及（4）捕获和稳定多蛋白复合物的 sAB。这些独特的功能为研究传统亲和试剂技术无法企及的复杂大分子过程提供了无数机会。我们提供了从头分离结合物的详细方案，以及下游生物物理特性分析的实例。所描述的方法具有通用性，可以适应基于酵母或 mRNA 展示的其他体外直接进化方法。
