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An open library of human kinase domain constructs for automated bacterial expression

Daniel L. Parton, Sonya M. Hanson, Lucelenie Rodríguez-Laureano, Steven K. Albanese, Scott Gradia, Chris Jeans, Markus Seeliger, Nicholas Levinson, John D. Chodera
doi: https://doi.org/10.1101/038711
Daniel L. Parton
1Computational Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065
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Sonya M. Hanson
1Computational Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065
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Lucelenie Rodríguez-Laureano
1Computational Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065
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Steven K. Albanese
2Gerstner Sloan Kettering Graduate School, Memorial Sloan Kettering Cancer Center, New York, NY 10065
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Scott Gradia
3QB3 MacroLab, University of California, Berkeley, CA 94720
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Chris Jeans
3QB3 MacroLab, University of California, Berkeley, CA 94720
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Markus Seeliger
4Department of Pharmacological Sciences, Stony Brook University Medical School, Stony Brook, NY 11794
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Nicholas Levinson
5Department of Pharmacology, University of Minnesota, Minneapolis, MN 55455
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John D. Chodera
1Computational Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065
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  • For correspondence: john.chodera@choderalab.org
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Abstract

Kinases play a critical role in cellular signaling pathways. Human kinase dysregulation has been linked to a number of diseases, such as cancer, diabetes, and inflammation, and as a result, much of the effort in developing treatments (and perhaps 30%of all current drug development effort) has focused on shutting down aberrant kinases with targeted inhibitors. While insect and mammalian expression systems are frequently utilized for the expression of human kinases, they cannot compete with the simplicity and cost-effectiveness of bacterial expression systems, which historically had found human kinases difficult to express. Following the demonstration that phosphatase coexpression could give high yields of Src and Abl kinase domains in inexpensive bacterial expression systems [1], we have performed a large-scale expression screen to generate a library of His-tagged human kinase domain constructs that express well in a simple automated bacterial expression system where phosphatase coexpression (YopH for Tyr kinases, lambda for Ser/Thr kinases) is used. Starting from 96 kinases with crystal structures and any reported bacterial expression, we engineered a library of human kinase domain constructs and screened their coexpression with phosphatase, finding 52 kinases with yields greater than 2 mg/L culture. All sequences and expression data are provided online at https://github.com/choderalab/kinase-ecoli-expression-panel, and the plasmids are in the process of being made available through AddGene.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted February 25, 2016.
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An open library of human kinase domain constructs for automated bacterial expression
Daniel L. Parton, Sonya M. Hanson, Lucelenie Rodríguez-Laureano, Steven K. Albanese, Scott Gradia, Chris Jeans, Markus Seeliger, Nicholas Levinson, John D. Chodera
bioRxiv 038711; doi: https://doi.org/10.1101/038711
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An open library of human kinase domain constructs for automated bacterial expression
Daniel L. Parton, Sonya M. Hanson, Lucelenie Rodríguez-Laureano, Steven K. Albanese, Scott Gradia, Chris Jeans, Markus Seeliger, Nicholas Levinson, John D. Chodera
bioRxiv 038711; doi: https://doi.org/10.1101/038711

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