ArticleProtein expression and purification2025
Purification and characterization of full-length monomeric TEC family kinase, ITK.
Article in Protein expression and purification, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. An erratum has been issued. Cited by 3 papers.
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Who cites it
3 citing papers in PubMed.
- LAT condensation gates PLCγ1 activation via bimodal LAT phosphorylation.Proceedings of the National Academy of Sciences of the United States of America · 2026Article
- Weak SLP-76/PLC-γ1 interaction fine-tunes T cell receptor signal strength to optimize T cell responsiveness.Nature communications · 2026Article
- Weak SLP-76-PLC-γ1 interaction in the LAT-nucleated multi-protein complex fine-tunes TCR signal strength to optimize T cell responsiveness.bioRxiv : the preprint server for biology · 2025Article
Corrections and comments
- Erratum issued
Authors and funding
7 authors.
Funding
Abstract
An early step in the activation of T cells via the T cell antigen receptor is the phosphorylation and activation of phospholipase C-γ1 (PLC-γ1) by the TEC family tyrosine kinase, interleukin-2 (IL-2) inducible T cell kinase (ITK). PLC-γ1 activation occurs within a multi-protein complex comprised of the enzymes ITK, PLC-γ1, and VAV, and the adapter molecules, LAT, Gads, SLP-76, and NCK. Studies of ITK activation and the role of this heptameric complex in regulating ITK activation and function have not been possible due to the lack of success in the expression and purification of full-length, monomeric ITK protein. In this study, we have produced soluble full-length wild-type ITK protein by co-expressing an N-terminal solubility-tagged ITK construct with a kinase-specific co-chaperone CDC37 in an insect cell line. Although the majority of the purified ITK protein is oligomerized, there is a 13-fold increase in the yield of monomeric protein production compared to the last reported purification. Previous studies suggest that the ITK oligomerization is mediated by intermolecular interactions. We created several mutants to disrupt these self-associations. Expression of one of these, the C96E/T110I mutant, produced 20 times more monomer than the wild-type construct. The in vitro characterization of these protein constructs showed that the purified protein is stable and functional. This successful purification and in vitro characterization of full-length monomeric ITK protein will aid in understanding the mechanism by which ITK is recruited into the heptameric complex and is enabled to phosphorylate and activate PLC-γ1.
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