Derewenda Zygmunt S
Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
Methods. 2004 Nov;34(3):354-63. doi: 10.1016/j.ymeth.2004.03.024.
Recombinant techniques are routinely used for the preparation of protein samples for structural studies including X-ray crystallography. Among other benefits, these methods allow for a vast increase in the amount of obtained protein as compared to purification from source tissues, ease of purification when fusion proteins containing affinity tags are used, introduction of SeMet for phasing, and the opportunity to modify the protein to enhance its crystallizability. Protein engineering may involve removal of flexible regions including termini and interior loops, as well as replacement of residues that affect solubility. Moreover, modification of the protein surface to induce crystal growth may include rational engineering of surface patches that can readily mediate crystal contacts. The latter approach can be used to obtain proteins of crystals recalcitrant to crystallization or to obtain well-diffracting crystals in lieu of wild-type crystals yielding data to limited resolution. This review discusses recent advances in the field and describes a number of examples of diverse protein engineering techniques used in crystallographic investigations.
重组技术通常用于制备用于结构研究（包括X射线晶体学）的蛋白质样品。与从源组织中纯化相比，这些方法具有诸多优势，包括可大量增加获得的蛋白质数量、使用含亲和标签的融合蛋白时易于纯化、引入硒代蛋氨酸用于相位分析，以及有机会对蛋白质进行修饰以提高其结晶性。蛋白质工程可能涉及去除包括末端和内部环在内的柔性区域，以及替换影响溶解性的残基。此外，对蛋白质表面进行修饰以诱导晶体生长可能包括对易于介导晶体接触的表面区域进行合理工程设计。后一种方法可用于获得难以结晶的晶体蛋白，或获得能产生高分辨率数据的高质量衍射晶体，以替代只能产生低分辨率数据的野生型晶体。本综述讨论了该领域的最新进展，并描述了晶体学研究中使用的多种蛋白质工程技术的一些实例。
