Microfluidics-Based Strategies for Protein Crystallography

Citation

Anderson, Megan Jo (2009) Microfluidics-Based Strategies for Protein Crystallography. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/P5ZD-0S21. https://resolver.caltech.edu/CaltechETD:etd-10172008-222221

Abstract

Protein crystallography is an invaluable tool for the study of biological processes at the molecular level. While several crystallization techniques are actively pursued in both academic and industrial laboratories to produce high-quality protein crystals, the use of microfluidic technology for structural biology was previously shown to improve protein crystallization over more traditional methods. This thesis describes a microfluidics-based crystallization strategy that was developed to increase the success rate of crystallizing challenging proteins. The crystallization strategy involves using multiple microfluidic devices to characterize the solubility trends of the crystallization target, to perform nanoliter volume free interface diffusion crystallization experiments designed around the solubility trends, and to enable in situ diffraction analysis of crystals grown in microfluidic devices. The crystallization strategy was applied to the crystallization of a dozen challenging proteins and increased the overall crystallization and diffraction success rates compared with conventional automation. The crystallization strategy was also utilized to crystallize four metabolic proteins and provides the first demonstration of in situ structure determination for novel crystallization targets using a microfluidic crystallization platform. Additional technological advances were accomplished by the development of a novel microfluidic device designed to address the specific challenges of membrane protein crystallography. To date, this microfluidic crystallization strategy has produced four novel protein structures and holds great promise for future work in the field of protein crystallography.

Thesis Files