| Autor: |
Rashmi Puja, Ayon Chakraborty, Shubhankar Dutta, Kakoli Bose |
| Jazyk: |
angličtina |
| Rok vydání: |
2022 |
| Předmět: |
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| Zdroj: |
MethodsX, Vol 9, Iss , Pp 101704- (2022) |
| Druh dokumentu: |
article |
| ISSN: |
2215-0161 |
| DOI: |
10.1016/j.mex.2022.101704 |
| Popis: |
Vaccinia-related kinases (VRK) are serine-threonine kinases that regulate several signaling pathways. The isoform-VRK2A of one such kinase VRK2 controls cell stress response by interacting with TAK1, a mitogen-activated protein 3 kinase (MAP3K), via its partly cytosolic C-terminal transmembrane domain (VTMD). To establish the driving force and identify the key residues of the VRK2A-TAK1 interaction, we expressed and purified the standalone 3.6 kDa VTMD in the bacterial system using a unique and atypical two-step approach, when the effort to obtain full-length VRK2A remained unsuccessful. Characterization of biophysical properties demonstrated that VTMD domain maintains its structural integrity. Furthermore, dissecting the VRK2A-TAK1 binding interface using in silico tools provided important cues toward engineering the VRK2A-TAK1 interface to modulate its functions with desired characteristics. Most importantly, this novel purification strategy demonstrates its universal applicability in protein biochemistry research by serving as a model system for obtaining difficult-to-purify small proteins or domains. • VRK2A is a highly disordered transmembrane (TM) kinase, whose TM domain interacts with TAK1 (transforming growth factor-β-activated kinase). • The standalone VRK2A-TM domain (VTMD) was purified using affinity chromatography followed by two-step centricon based approach. • Biophysical and in silico analyses confirmed structural integrity of the domain. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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