Ultra-micro Amount And High Throughput Microfluidic Techniques For Protein Crystallization And Screening

Currently, X-ray crystallography is the most important and reliable method for protein structural determination. The production of diffraction-quality crystals is still the major bottleneck in the application of protein crystallography to large-scale structural biology research. Since there is no theoretical way to predict the suitable crystallization condition, current protein crystallization study is mainly based on trial and error approach. Large-scale screening of hundreds, or even thousands of chemical conditions involving various salts, buffers, and precipitating agents against one protein target is necessary to search for the right crystallization condition. Such a screening requires large amount of protein at milligram level, and thus is unrealistic for many scarce protein targets. With the advantages of high throughput, low consumption and large-scale integration, microfluidics has proven to be a promising technique for protein crystallization and screening.In Chapter 1, the methodology, advantages and limitations of three major methods in the current protein structural study are briefly introduced. We review the recent progress of various microfluidic techniques successfully applied in protein crystallization, including PDMS chip-based valve and pump, droplet, SlipChip, and DropLab. In addition, due to the high importance and great challenges of membrane proteins in structural biology, we further introduce the microfluidic techniques for crystallization of membrane proteins.In Chapter 2, we developed an ultra-micro amount and high-throughput protein crystallization screening system based on sequential operation droplet array (SODA) technique. To improve the reliability and throughput of the SODA system for protein crystallization, we developed two novel droplet manipulation methods based on continuous reagent addition with fixed volume and variable volumes. To test the performance of the present system, we screened five soluble protein crystallization conditions using commercial kits containing 96 different precipitants. Each screening experiment was performed in an array of 288 droplets with a volume of 2.01 nL for each droplet. Thus, the total consumption of protein solution was less than 0.6 ?L, which is 2-3 orders of magnitude lower than those of conventional systems. We comprehensively studied the effects of the droplet volume on protein crystallization behavior in the range of picoliter to microliter. The present system was also used to produce a droplet array with linearly increased protein and precipitant concentrations to study the protein phase behavior. A 9× 9 droplet array with a droplet volume of 8.04 nL can be generated within 9 min, and the total consumption of protein solution for each phase diagram determination was significantly reduced to ca.200 nL. Finally, this system was preliminary applied to screen the crystallization condition of a membrane protein, mechanosensitive channel of large conductance (MscL).