1 research outputs found
Supplementary guidance: listening to staff: Autumn 2017
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