Sommer Morten O A, Larsen Sine
Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, Denmark.
J Synchrotron Radiat. 2005 Nov;12(Pt 6):779-85. doi: 10.1107/S0909049505002621. Epub 2005 Oct 18.
Crystallization of proteins from a purified protein solution remains a bottleneck in the structure determination pipeline. In this paper the crystallization problem is addressed using a microfluidic device capable of determining detailed protein precipitation diagrams using less than 10 microL of protein sample. Based on the experimentally determined protein phase behavior, a crystallization screen can be designed to accommodate the physical chemistry of the particular protein target. Such a tailor-made crystallization screen has a high probability of yielding crystallization hits. The approach is applied to two different proteins: the calcium pump (SERCA), an eukaryotic integral membrane protein, and UMP kinase, a prokaryotic soluble kinase. Protein phase behavior is mapped for both proteins and tailor-made crystallization screens are designed for the two proteins resulting in about 50% crystallization probability per experiment. This illustrates the power of using microfluidic devices for detailed characterization of protein phase behavior prior to crystallization trials.
从纯化的蛋白质溶液中结晶蛋白质仍然是结构测定流程中的一个瓶颈。在本文中，使用一种微流控装置来解决结晶问题，该装置能够用少于10微升的蛋白质样品确定详细的蛋白质沉淀图。基于实验确定的蛋白质相行为，可以设计一个结晶筛选方案来适应特定蛋白质靶点的物理化学性质。这样一个量身定制的结晶筛选方案有很高的概率产生结晶成功的结果。该方法应用于两种不同的蛋白质：钙泵（SERCA），一种真核整合膜蛋白，以及UMP激酶，一种原核可溶性激酶。绘制了这两种蛋白质的相行为图，并为这两种蛋白质设计了量身定制的结晶筛选方案，每次实验的结晶概率约为50%。这说明了在进行结晶试验之前，使用微流控装置详细表征蛋白质相行为的作用。
