High throughput Imaging System for analysis of protein crystals

生物医学研究越来越多地需要对相关靶蛋白的三维结构进行分析（例如，为了理解重要的病理机制，开发特定的小分子抑制剂或基于抗体的疗法）。这种靶蛋白的结构表征在很大程度上是通过蛋白质晶体的x射线晶体学分析来完成的。这种结构表征的主要瓶颈之一是合适的蛋白质晶体的生长，因此控制和优化结晶条件是成功的关键。这种优化通常基于使用自动成像系统对高通量结晶方法的分析，允许对结晶过程进行跟踪，以使蛋白质结晶步骤尽可能高效。因此，我们计划在莱尔贝克大学生物化学研究所安装这样一个成像系统。传统成像系统通常侧重于通过各种手段（例如，白光，紫外线）分析晶体生长，以检测甚至难以可视化的晶体（例如，脂质立方相中的微晶体或小膜蛋白晶体）。相比之下，所提出的系统提供了一个动态光散射模块，它有助于监测蛋白质溶液的单分散性，从而间接地监测蛋白质的稳定性，作为不同缓冲条件的函数。这种分析只需要少量的蛋白质（~50 nl），因此构成了任何高通量结晶策略的廉价和有用的附加。使用该方法进行的初步实验表明，这种优化大大减少了实际蛋白质结晶所需的高通量结晶实验次数，从而提高了结晶过程的效率。此外，动态光散射模块允许在各种条件下对成核和晶体生长进行精确的动力学分析，从而为结晶优化提供了另一个重要参数。总的来说，所要求的设备将通过有针对性地优化缓冲液和结晶条件，从而提高蛋白质结晶的效率，从而显著提高重组蛋白的使用效率。

Biomedical research requires to a growing extent the analysis of three-dimensional structures of relevant target proteins (e.g., for understanding important pathomechanisms, the development of specific small molecule inhibitors or antibody-based therapeutics). The structural characterization of such target proteins is to a large extent performed by X-ray crystallographic analysis of protein crystals. One of the main bottlenecks of such a structural characterization is the growth of suitable protein crystals and therefore the control and optimization of crystallization conditions is key to success. Such optimization is usually based on an analysis of high throughput crystallization approaches using an automated imaging system, allowing for the tracing of crystallization progress to render the protein crystallization step as efficient as possible. We therefore plan to install such an imaging system at the Institute of Biochemistry of the University of Lübeck.Conventional imaging systems typically focus on the analysis of crystal growth by various means (e.g., white light, ultraviolet) to detect even difficult to visualize crystals (e.g., microcrystals or small membrane protein crystals in lipidic cubic phase). In contrast, the proposed system offers a dynamic light scattering module, which facilitates to monitor also the monodispersity of a protein solution - and thus indirectly protein stability - as a function of different buffer conditions. This analysis requires only minimal amounts of the protein (~50 nl) and thus constitutes a cheap and useful add-on to any high-throughput crystallization strategy. Initial experiments using this method revealed that such an optimization significantly reduces the number of high-throughput crystallization experiments required for the actual protein crystallization and thus the crystallization process becomes more efficient. In addition, the dynamic light scattering module allows accurate kinetic analysis of nucleation and crystal growth under a variety of conditions, thus providing an additional important parameter for crystallization optimization. Overall, the requested device will thus lead to a significantly more efficient use of the recombinant protein by targeted optimization of both buffer and crystallization conditions and thus an increased efficiency of protein crystallization.