基于 slipChip 的多参数筛选用于纳升级蛋白质结晶的自由界面扩散和微批量方法。
Multiparameter screening on SlipChip used for nanoliter protein crystallization combining free interface diffusion and microbatch methods.
机构信息
Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
出版信息
J Am Chem Soc. 2010 Jan 13;132(1):112-9. doi: 10.1021/ja908558m.
This paper describes two SlipChip-based approaches to protein crystallization: a SlipChip-based free interface diffusion (FID) method and a SlipChip-based composite method that simultaneously performs microbatch and FID crystallization methods in a single device. The FID SlipChip was designed to screen multiple reagents, each at multiple diffusion equilibration times, and was validated by screening conditions for crystallization of two proteins, enoyl-CoA hydratase from Mycobacterium tuberculosis and dihydrofolate reductase/thymidylate synthase from Babesia bovis, against 48 different reagents at five different equilibration times each, consuming 12 microL of each protein for a total of 480 experiments using three SlipChips. The composite SlipChip was designed to screen multiple reagents, each at multiple mixing ratios and multiple equilibration times, and was validated by screening conditions for crystallization of two proteins, enoyl-CoA hydratase from Mycobacterium tuberculosis and dihydrofolate reductase/thymidylate synthase from Babesia bovis. To prevent cross-contamination while keeping the solution in the neck channels for FID stable, the plates of the SlipChip were etched with a pattern of nanowells. This nanopattern was used to increase the contact angle of aqueous solutions on the surface of the silanized glass. The composite SlipChip increased the number of successful crystallization conditions and identified more conditions for crystallization than separate FID and microbatch screenings. Crystallization experiments were scaled up in well plates using conditions identified during the SlipChip screenings, and X-ray diffraction data were obtained to yield the protein structure of dihydrofolate reductase/thymidylate synthase at 1.95 A resolution. This free-interface diffusion approach provides a convenient and high-throughput method of setting up gradients in microfluidic devices and may find additional applications in cell-based assays.
本文描述了两种基于 SlipChip 的蛋白质结晶方法：一种基于 SlipChip 的自由界面扩散（FID）方法和一种同时在单个设备中进行微批量和 FID 结晶方法的基于 SlipChip 的组合方法。FID SlipChip 被设计用于筛选多种试剂，每种试剂都有多个扩散平衡时间，并通过筛选两种蛋白质的结晶条件进行了验证，这两种蛋白质分别是结核分枝杆菌烯酰辅酶 A 水合酶和巴贝斯牛 Babesia bovis 二氢叶酸还原酶/胸苷酸合酶，针对 48 种不同的试剂，每种试剂有 5 个不同的平衡时间，总共使用三个 SlipChip 消耗了 12μL 的每种蛋白质，进行了 480 次实验。组合式 SlipChip 被设计用于筛选多种试剂，每种试剂都有多个混合比和多个平衡时间，并通过筛选两种蛋白质的结晶条件进行了验证，这两种蛋白质分别是结核分枝杆菌烯酰辅酶 A 水合酶和巴贝斯牛 Babesia bovis 二氢叶酸还原酶/胸苷酸合酶。为了防止交叉污染，同时保持 FID 颈通道中的溶液稳定，SlipChip 的板用纳米孔图案进行了蚀刻。这种纳米图案用于增加水溶液在硅烷化玻璃表面的接触角。组合式 SlipChip 增加了成功结晶条件的数量，并比单独的 FID 和微批量筛选确定了更多的结晶条件。使用 SlipChip 筛选过程中确定的条件在微孔板中放大了结晶实验，并获得了 X 射线衍射数据，以 1.95Å 的分辨率得到了二氢叶酸还原酶/胸苷酸合酶的蛋白质结构。这种自由界面扩散方法为在微流控设备中建立梯度提供了一种方便且高通量的方法，并且可能在基于细胞的测定中找到其他应用。
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