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Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins

Li, Liang and Mustafi, Debarshi and Fu, Qiang and Tereshko, Valentina and Chen, Delai L. and Tice, Joshua D. and Ismagilov, Rustem F. (2006) Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins. Proceedings of the National Academy of Sciences of the United States of America, 103 (51). pp. 19243-19248. ISSN 0027-8424. PMCID PMC1748211. https://resolver.caltech.edu/CaltechAUTHORS:20130821-160724073

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Abstract

High-throughput screening and optimization experiments are critical to a number of fields, including chemistry and structural and molecular biology. The separation of these two steps may introduce false negatives and a time delay between initial screening and subsequent optimization. Although a hybrid method combining both steps may address these problems, miniaturization is required to minimize sample consumption. This article reports a "hybrid" droplet-based microfluidic approach that combines the steps of screening and optimization into one simple experiment and uses nanoliter-sized plugs to minimize sample consumption. Many distinct reagents were sequentially introduced as approx. 140-nl plugs into a microfluidic device and combined with a substrate and a diluting buffer. Tests were conducted in approx. 10-nl plugs containing different concentrations of a reagent. Methods were developed to form plugs of controlled concentrations, index concentrations, and incubate thousands of plugs inexpensively and without evaporation. To validate the hybrid method and demonstrate its applicability to challenging problems, crystallization of model membrane proteins and handling of solutions of detergents and viscous precipitants were demonstrated. By using 10 μl of protein solution, approx. 1,300 crystallization trials were set up within 20 min by one researcher. This method was compatible with growth, manipulation, and extraction of high-quality crystals of membrane proteins, demonstrated by obtaining high-resolution diffraction images and solving a crystal structure. This robust method requires inexpensive equipment and supplies, should be especially suitable for use in individual laboratories, and could find applications in a number of areas that require chemical, biochemical, and biological screening and optimization.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doiorg/10.1073/pnas.0607502103DOIArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/pmc1748211/PubMed CentralArticle
http://www.pnas.org/content/103/51/19243/suppl/DC1PublisherSupporting Information
ORCID:
AuthorORCID
Ismagilov, Rustem F.0000-0002-3680-4399
Additional Information:© 2006 by The National Academy of Sciences of the USA. Edited by Robert M. Stroud, University of California, San Francisco, CA, and approved October 27, 2006 (received for review August 29, 2006). We thank Nina Ponomarenko and James R. Norris [University of Chicago (UC)] for samples of RC from R. viridis, the Philip D. Laible group at Argonne National Laboratory (ANL), UC/ANL Collaborative Seed Funding for samples of Porin R. capsulatus, Cory J. Gerdts for the image in Fig. 2 c and for helpful discussions, and Jessica Price for contributions in editing and writing this manuscript. Use of the ANL Structural Biology Center beamlines, BioCARS beamlines, and the National Institute for General Medical Sciences (GM) and National Cancer Institute (CA) GM/CA beamlines at the Advanced Photon Source was supported by Department of Energy Grant W-31-109-Eng-38. GM/CA-Collaborative Access Team (CAT) has been funded in whole or in part by National Cancer Institute Grant Y1-CO-1020 and National Institute of General Medical Sciences (NIGMS) Grant Y1-GM-1104. Use of the BioCARS Sector 14 was supported by National Institutes of Health (NIH) National Center for Research Resources (NCRR) Grant RR07707. We thank ATCG3D funded by the NIGMS and NCRR under the PSI-2 Specialized Center program (U54 GM074961) for partial support of V.T. and deCode Biostructures for providing the custom screening kit for membrane protein crystallization. Undergraduate research was supported by the NIH Roadmap Physical and Chemical Biology training program at UC (D.M.). This work was supported in part by NIH Roadmap for Medical Research Grant R01 GM075827-01). Author contributions: L.L., D.M., Q.F., D.L.C., J.D.T., and R.F.I. designed research; L.L., D.M., Q.F., D.L.C., and J.D.T. performed research; L.L., D.M., Q.F., D.L.C., and J.D.T. contributed new reagents/analytic tools; L.L., D.M., Q.F., V.T., D.L.C., J.D.T., and R.F.I. analyzed data; and L.L., D.M., Q.F., V.T., and R.F.I. wrote the paper. The authors declare no conflict of interest. This article is a PNAS direct submission. Data deposition: The coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 2I5N).
Funders:
Funding AgencyGrant Number
Department of Energy (DOE)W-31-109-ENG-38
NIHY1-CO-1020
NIHY1-GM-1104
NIHRR07707
NIHU54 GM074961
NIHR01 GM075827-01
National Cancer InstituteUNSPECIFIED
National Institute of General Medical SciencesUNSPECIFIED
Issue or Number:51
PubMed Central ID:PMC1748211
Record Number:CaltechAUTHORS:20130821-160724073
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20130821-160724073
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:40825
Collection:CaltechAUTHORS
Deposited By: Whitney Barlow
Deposited On:27 Aug 2013 23:40
Last Modified:03 Jun 2020 20:06

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