Multiparameter Screening on SlipChip Used for Nanoliter Protein Crystallization Combining Free Interface Diffusion and Microbatch Methods

摘要

This paper describes two SlipChip-based approaches to protein crystallization: a SlipChip-basedudfree interface diffusion (FID) method and a SlipChip-based composite method that simultaneously performsudmicrobatch and FID crystallization methods in a single device. The FID SlipChip was designed to screenudmultiple reagents, each at multiple diffusion equilibration times, and was validated by screening conditionsudfor crystallization of two proteins, enoyl-CoA hydratase from Mycobacterium tuberculosis and dihydrofolateudreductase/thymidylate synthase from Babesia bovis, against 48 different reagents at five different equilibrationudtimes each, consuming 12 µL of each protein for a total of 480 experiments using three SlipChips. Theudcomposite SlipChip was designed to screen multiple reagents, each at multiple mixing ratios and multipleudequilibration times, and was validated by screening conditions for crystallization of two proteins, enoylCoA hydratase from Mycobacterium tuberculosis and dihydrofolate reductase/thymidylate synthase fromudBabesia bovis. To prevent cross-contamination while keeping the solution in the neck channels for FIDudstable, the plates of the SlipChip were etched with a pattern of nanowells. This nanopattern was used toudincrease the contact angle of aqueous solutions on the surface of the silanized glass. The composite SlipChipudincreased the number of successful crystallization conditions and identified more conditions for crystallizationudthan separate FID and microbatch screenings. Crystallization experiments were scaled up in well platesudusing conditions identified during the SlipChip screenings, and X-ray diffraction data were obtained to yieldudthe protein structure of dihydrofolate reductase/thymidylate synthase at 1.95 Å resolution. This free-interfaceuddiffusion approach provides a convenient and high-throughput method of setting up gradients in microfluidicuddevices and may find additional applications in cell-based assays.