STAR Protocols
Volume 2, Issue 1, 19 March 2021, 100376
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Protocol
Expression and purification of fused kinase from insect cells for in vitro kinase assay

https://doi.org/10.1016/j.xpro.2021.100376Get rights and content
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Highlights

  • Purification of constitutively active Fu from insect cells for in vitro kinase assay

  • Priming phosphorylation by Fu can allow secondary in vitro kinase assay

  • High-purity protein elution with Flag M2 affinity agarose and 3X Flag peptide

  • Sensitive phospho-protein detection via pIMAGO-biotin kit or specific antibodies

Summary

The Fused (Fu) kinase is a key transducer of Hedgehog signaling, but its relevant substrates have remained obscured due to the difficulty of obtaining active Fu for in vitro kinase assay. Based on the mechanism of Fu activation in vivo, we engineered a constitutively active Fu and expressed it in Sf9 cells using the baculovirus system. The kinase was affinity purified and applied for in vitro kinase assay using recombinant GST-fusion proteins as substrates to identify Fu-specific phosphorylation sites.

For complete details on the use and execution of this protocol, please refer to Han et al. (2019).

Subject areas

Molecular biology
Protein biochemistry
Protein expression and purification

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