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

Biochemistry
Steven K AlbaneseJohn D Chodera

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...Continue Reading

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Citations

Feb 23, 2021·The Journal of Biological Chemistry·Manas Pratim ChakrabortyArindam Mukherjee
Apr 2, 2021·The Journal of Clinical Investigation·Qian ZhangThomas L Clemens
Oct 30, 2020·Biophysical Journal·Robert B Best
Sep 22, 2021·The Journal of Cell Biology·Carolyn M KellyJohn P O'Donnell
Nov 10, 2021·Proceedings of the National Academy of Sciences of the United States of America·Agatha LyczekMarkus A Seeliger

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