Accelerated Protein Crystallization with FID

Courtesy of Fluidigm Remember science class when you had to grow salt or sugar crystals? Remember how some people would end up with fabulous crystals, while others would end up with--well, not so fabulous crystals, all because the conditions in each glass of water were slightly different? Now imagine doing that with proteins. Of course, no one uses strings and a glass of supersaturated water, but the concept is basically the same. In protein crystallization, however, the solute is often expensive or scarce. And protein crystals can form under a variety of conditions; finding the right one is often the challenge. The two most popular methods for protein crystallization are microbatch and vapor diffusion. For both methods, the protein and reagent are combined in fixed ratios. In the microbatch method, the solution is incubated under a layer of immiscible oil, and in vapor diffusion, the solution is allowed to slowly dehydrate. Now, there's a third option. the Topaz™ Crystallizer, newly developed and released by Fluidigm of South San Francisco, Calif., uses disposable fluidic microprocessors that allow free interface diffusion (FID). Fluidigm claims this technology is more efficient than other methods--requiring less purified protein--and that it also generates protein crystals faster and more cost effectively. FID is a process in which a low-density solution is layered onto a solution of higher density. The two fluids are allowed to mix by direct contact and to reach equilibrium gradually. FID has not been widely used because it requires the use of capillary tubes. "It's difficult to set up in capillary tubes," explains product manager Kristin Spataro. "A lot of times you get a lot of convection. This is a gentler form of crystal formation." The instrument is designed so that the protein and reagents meet and diffuse in a narrow channel, allowing equilibrium to occur slowly and eliminating convection currents. "As soon as you open the interface valve, which separates the protein side and the reagent, there's interface. Right there the protein sees the reagent," explains Spataro. As little as 3 µl of the protein is needed for simultaneous screening with 48 different reagents. Each of the 48 metering cells on the microprocessor allows for three different concentration ratios--4:1, 1:1, and 1:4--for 144 reactions. "There's two orders of magnitude savings there," says Spataro. "That's the area that has resonated well with [users]." Currently, the device supports two chips at once, but Fluidigm is working on increasing the throughput. The company's technical support team can help labs set up massive parallel-screening systems, for 40 tandem microprocessors, for instance. One technician could then screen 5,760 unique conditions in one week using only 120 µl of protein. LOW VOLUME, BIG PLUS "For us the low volume is a big plus," says Peter Nollert, senior research scientist at Reykjavik, Iceland-based deCODE Genetics' Biostructures Group. "It can take months or years to establish a purified product. At the outset you don't know what conditions give you crystals. You need to set up as many environments as possible." But for proteins that cannot be obtained in large quantities or those that are expensive to produce, sometimes only 50 to 100 crystallization experiments can be set up. Nollert says deCODE is interested in membrane proteins, but these proteins are expensive to make, so a microprocessor that requires less product makes a big difference. "To give it a good crack you want to set up several thousand experiments. This [Topaz Crystallizer] really helps us in pursuing difficult targets," Nollert says. Spataro says Fluidigm has 12 customers for the Topaz Crystallizer and expects a couple more in the next few weeks. Pharmaceutical and biotech companies, as well as academic labs have expressed interest, which has increased since the publication of a study reporting that crystals grow faster using the Topaz Crystallizer than other methods.1 In some cases, crystals were observed after only a few hours. The paper also reported that the Topaz Crystallizer was able to generate protein crystals that had not been observed using conventional methods. Although the Topaz Crystallizer is on the market, it is still early in its development. Spataro didn't want to go into details about what additions or improvements would be made to the system, but she did say that a complete system is expected by 2004. --Christine Yanicek 1. C.L. Hansen et al., "A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion," Proc Natl Acad Sci, 99:16531-6, Dec. 24, 2002.

Accelerated Protein Crystallization with FID

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