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Biochemistry. 2018 Aug 7;57(31):4675-4689. doi: 10.1021/acs.biochem.7b01081. Epub 2018 Jul 26.

An Open Library of Human Kinase Domain Constructs for Automated Bacterial Expression.

Author information

1
Louis V. Gerstner, Jr Graduate School of Biomedical Sciences , Memorial Sloan Kettering Cancer Center , New York , New York 10065 , United States.
2
Computational and Systems Biology Program, Sloan Kettering Institute , Memorial Sloan Kettering Cancer Center , New York , New York 10065 , United States.
3
Tri-Institutional PhD Program in Chemical Biology, Weill Cornell Graduate School of Medical Sciences , Cornell University , New York , New York 10065 , United States.
4
Tri-Institutional Program in Computational Biology and Medicine, Weill Cornell Graduate School of Medical Sciences , Cornell University , New York , New York 10065 , United States.
5
QB3MacroLab , University of California , Berkeley , California 94720 , United States.
6
Department of Pharmacology , University of Minnesota , Minneapolis , Minnesota 55455 , United States.
7
Department of Pharmacological Sciences , Stony Brook University Medical School , Stony Brook , New York 11794 , United States.

Abstract

Kinases play a critical role in cellular signaling and are dysregulated in a number of diseases, such as cancer, diabetes, and neurodegeneration. Therapeutics targeting kinases currently account for roughly 50% of cancer drug discovery efforts. The ability to explore human kinase biochemistry and biophysics in the laboratory is essential to designing selective inhibitors and studying drug resistance. Bacterial expression systems are superior to insect or mammalian cells in terms of simplicity and cost effectiveness but have historically struggled with human kinase expression. Following the discovery that phosphatase coexpression produced high yields of Src and Abl kinase domains in bacteria, we have generated a library of 52 His-tagged human kinase domain constructs that express above 2 μg/mL of culture in an automated bacterial expression system utilizing phosphatase coexpression (YopH for Tyr kinases and lambda for Ser/Thr kinases). Here, we report a structural bioinformatics approach to identifying kinase domain constructs previously expressed in bacteria and likely to express well in our protocol, experiments demonstrating our simple construct selection strategy selects constructs with good expression yields in a test of 84 potential kinase domain boundaries for Abl, and yields from a high-throughput expression screen of 96 human kinase constructs. Using a fluorescence-based thermostability assay and a fluorescent ATP-competitive inhibitor, we show that the highest-expressing kinases are folded and have well-formed ATP binding sites. We also demonstrate that these constructs can enable characterization of clinical mutations by expressing a panel of 48 Src and 46 Abl mutations. The wild-type kinase construct library is available publicly via Addgene.

PMID:
30004690
PMCID:
PMC6081246
[Available on 2019-08-07]
DOI:
10.1021/acs.biochem.7b01081

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