11 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
Data collection and refinement statistics for methylglyoxal reductase (YghZ).
<p>*Values in parentheses are for highest-resolution shell.</p
Crystallization of native source <i>E. coli</i> proteins.
<p>(A) Capillary electrophoresis of purified protein fractions. White stars indicate samples successfully crystallized and black stars represent solved structures. (B) Crystals of 5-keto-4-deoxyuronate isomerase crystallized from fractions of varying purity. Crystal quality was not always correlated with sample purity. (C) Resolution of the data collected versus percent purity of the starting sample based on quantification of protein concentrations by capillary gel electrophoresis with the Caliper system. Sample purity did not correlate with higher resolution data.</p
Proteome fractionation and purification flow chart.
<p>Approximately 500 g of <i>E. coli</i> cells were lysed at pH 7 using a microfluidizer and the cell debris pelleted. The supernatant was applied to a tangential flow column with a nominal molecular weight cut off of 500 kDa, generating 2 fractions (retentate and flow through). The fraction above 500 kDa (retentate) was further purified via sucrose gradients, size exclusion, and ion exchange chromatography prior to crystallization trials. The fraction less than 500 kDa was applied to multiple affinity and ion exchange columns followed by phenyl sepharose, ion exchange, and size exclusion prior to crystallization trials in microfluidic chips.</p
Data collection and refinement statistics for Glucose-6-phosphate isomerase (pGI).
<p>*Values in parentheses are for highest-resolution shell.</p
Crystallization conditions and data collection statistics for previously deposited structures.
<p>Crystallization conditions and data collection statistics for previously deposited structures.</p
Data collection and refinement statistics for glutamate dehydrogenase (GDH).
<p>Data collection and refinement statistics for glutamate dehydrogenase (GDH).</p
BglA dimer and putative active site.
<p>Left, BglA dimer with the putative active site outlined in a gray box. Right, close up of the active site with glucose-6-phosphate modeled based of the position of the sulfate ion from crystallization. Active site residues are depicted as ball-and-stick. Putative hydrogen bonds to the substrate are drawn as dashed lines.</p
Data collection and refinement statistics for 6-phospho-beta-glucosidase (BglA).
<p>*Values in parentheses are for highest-resolution shell.</p
GDH hexamer from <i>E. coli</i>.
<p>The protein forms a hexamer (dimer of trimers). Left, view of the GDH hexamer along the two-fold axis. Right, view of the GDH hexamer along the three-fold axis.</p