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

• DOI: 10.1021/acs.biochem.7b01081
• Publication Date: 2018-07-13T18:29:35Z
• Authors: Steven K. Albanese; Daniel L. Parton; Mehtap Işık; Lucelenie Rodrígu...; Sonya M. Hanson; Julie M. Behr; Scott Gradia; Chris Jeans; Nicholas M. Levinson; Markus A. Seeliger; John D. Chodera
• Keywords: Protein kinase domain; ABL

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