Microfluidics for Protein Crystallization

Brandeis University, Waltham MA

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Abstract

DESCRIPTION (provided by applicant): Protein crystals are necessary in order to determine protein structure using x-ray diffraction. Typically the number of crystallization trials are limited by the availability of protein, hence the drive to minimize sample volume. To address this problem a high-throughput, low volume microfluidic device denoted the phase chip will be developed. On this device different microfluidic components will be designed, fabricated, and interconnected in order to precisely meter, mix, and store subnanoliter amounts of sample, solvent, and other reagents. The phase chip can store thousands of sub-nanoliter drops of protein solution in individual wells and a total of 10-3 crystallization trials can be accomplished with 1 -10 u.g of protein thereby enabling high-throughput crystallization of mammalian proteins expressed in tissue culture. Additionally each sample well is in contact with a reservoir through a dialysis membrane through which only water and other low molecular weight organic solvents can pass. Thus the concentration of all solutes in an aqueous solution can be reversibly, rapidly, and precisely varied in contrast to current microfluidic crystallization methods, which are irreversible. Rapid reversible dialysis solves a major problem in protein crystallization, the decoupling of nucleation from growth. Using the phase chip we will screen crystallization conditions using proteins that are not available in sufficient quantities for current techniques. The protein targets are bacterially-expressed recombinant channel proteins, G protein-coupled receptors heterologously expressed in a mammalian cell culture system, and enzymes which produce crystals too small for diffraction.