Abstract
The lack of high-throughput approaches for expression and screening of large enzyme libraries remains a major bottleneck for current enzyme engineering efforts. To address this need, we have developed a high-throughput, fluorescence-based approach for rapid one-pot, microscale expression, and screening of industrial enzymes. In this chapter, we present the protocol for integration of cell-free protein expression with activity screening of enzymes in two formats: (1) a 96-well plate format and (2) a microscale-array format. Our one-pot method is ideally suited for rapid, first pass screening of enzymes and can also be used to perform detailed mechanistic analysis such as measurement of kinetics, determination of optimum temperature, and to study enzyme inhibition.
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Acknowledgments
The authors would like to thank Dr. Joshua I. Park for assistance with Gateway cloning techniques and Dr. Chung-Yan Koh for critical reading of the manuscript. This work was part of the DOE Joint BioEnergy Institute (http://www.jbei.org) supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy. Sandia is a multiprogram laboratory operated by Sandia Corp., a Lockheed Martin Co., for the US Department of Energy under Contract DE-AC04-94AL85000.
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Chandrasekaran, A., Singh, A.K. (2014). One-Pot, Microscale Cell-Free Enzyme Expression and Screening. In: Alexandrov, K., Johnston, W. (eds) Cell-Free Protein Synthesis. Methods in Molecular Biology, vol 1118. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-782-2_4
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DOI: https://doi.org/10.1007/978-1-62703-782-2_4
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Publisher Name: Humana Press, Totowa, NJ
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