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

UID: 10382

Description
Source code, assay data, full expression results for all ABL1 constructs, wild-type kinases, and ABL1 and SRC mutants, plasmid libraries, and other supporting material for the generation of a library of human kinase domain constructs for automated bacterial expression. From the publication 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."
Subject of Study
Subject(s)
Access via GitHub

Chodera Lab github repository containing expression data, database construction code, plasmid libraries, assay data, interactive table of expression data, and other project files.

Access via Addgene

Human Kinase Domain Constructs Kit

Access Restrictions
Free to All
Fee Required
Access Instructions
The public Github repo for the Chodera lab's kinase ecoli expression panel contains data, code and other project specific files including an interactive construct expression browser hosted on github.io. The link to the expression browser is located in the README.md. The Human Kinase Domain Contructs Kit contains 68 human kinase catalytic domain constructs and the respective phosphatases that enhance their bacterial expression for purchase through addgene although the individual plasmid information (backbone, gene/insert, partial sequence, etc) is freely available. To get to the individual plasmid data, go to 'Contents of the Kit' then select the plasmid from the inventory list or plate map. The site also includes ordering information for individual plasmids.
Associated Publications
Dataset Format(s)
PDF, Microsoft Excel, CSV, Plain Text, HTML, XML, tar, YAML, Python