Microfluidic platforms for protein crystallization and in situ X-ray analysis

microfluidics;protein crystallization;structure determination

Guha, Sudipto ; Kenis ; Paul J.A.

关键词: microfluidics;    protein crystallization;    structure determination;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/18620/Guha_Sudipto.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Recent efforts associated with the Protein Structure Initiative from the National Institutes of Health (NIH) have eased the bottlenecks associated with protein expression, purification, and crystallization for structural analysis.While strategies have been developed for dealing with each of these challenges in a high throughput fashion, crystal harvesting and mounting for X-ray structure determination is still largely a manual process.During mounting there is a significant potential for damaging fragile protein crystals, both from physical and environmental shock.Microfluidic strategies for protein crystallization have been limited in their application because of a variety of reasons including (i) difficulties in harvesting crystals from chips and (ii) translating results obtained on-chip to a larger scale.The fabrication and operation of an X-ray compatible microfluidic device architecture that allows for on-chip crystallization and in situ X-ray analysis of protein crystals while maintaining the advanced fluid handling capabilities of multilayer microfluidics is reported here.The design uses a traditional thin polydimethylsiloxane (PDMS) fluid layer to enable the actuation of pneumatic valves.The traditional glass substrate and thick PDMS control layer are replaced with thin layers of cyclic olefin copolymer (COC).This hybrid COC-PDMS device architecture takes advantage of the low X-ray scattering cross-section of organic polymers such as COC while minimizing the path-length of the more strongly scattering silicon-based PDMS.The device also addresses concerns associated with evaporative losses from the traditional PDMS based devices.X-ray compatible microfluidic chips consisting of arrays of well were implemented using this architecture.The quality of crystals grown on-chip can be screened in situ and either standard single-crystal cryogenic analysis or room temperature "single-shot" analysis of many crystals can be used to collect data for protein structure determination.We validated our approach by crystallizing the soluble proteins lysozyme, thaumatin, ribonuclease A, bacterial lyase and bacterial defluorinase on-chip and performing in situ X-ray crystallographic analysis.