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Technology Description	This paper describes a method of time-controlled seeding to separate the stages of nucleation and growth in protein crystallization using a microfluidic device. We quantified microfluidic seeding using a model protein, developed a strategy to produce diffraction-quality crystals of proteins recalcitrant to traditional methods, and solved de novo the X-ray crystal structure of Oligoendopeptidase F.
Category	Structure
PRC	Scripps Research Institute
PubMed ID	17099920
Author	Cory J. Gerdts, Valentina Tereshko, Maneesh K. Yadav, Irina Dementieva, Frank Collart, Andrzej Joachimiak, Raymond C. Stevens, Peter Kuhn, Anthony Kossiakoff, and Rustem F. Ismagilov
Publication Description	Proteins are crystallized to determine their three-dimensional structures, to understand protein function, and aid in drug design, but crystallization can be an unpredictable and stochastic process. Microfluidics is emerging as a tool to perform crystallization trials faster, cheaper, in smaller volumes, and with a higher level of control.
Methodology	The system uses soft lithography microfluidics to form plugs of controlled size and composition surrounded by a fluorocarbon carrier fluid in glass microcapillaries. To separate the nucleation and growth stages, a microfluidic device was designed to control multiple reactions in sequence and in time. To validate this method with challenging structurally uncharacterized targets, we crystallized SARS nucleocapsid N-terminal domain (“SARS protein”) and Oligoendopeptidase F from Bacillus stearothermophilus.

National Institute of Allergy and Infectious Disease