We offer the plant research community and companies access to our protein complex purification platform1,2,3.
Purifications under research service agreement are performed based on well-established protocols for complex purification from either the Arabidopsis cell suspension cultures PSB-D/L, seedlings or isolated plant tissues. We have recently been able to show that our complex purification technology performs equally well in crop species such as corn4,5 and rice6. We have extensive experience with tandem affinity purification (TAP) for the isolation of stable protein complexes at high purity, whereas for more short-term interactions, we apply pull-downs using GFP-containing tags, or a more efficient protocol based on the Protein A/G moiety present in our TAP tags. More recently, we have developed an approach to enrich for unstable protein interactions by means of protein cross-linking.
Schematic Overview of the GSrhino-based Pull-Down and TAP Protocols.
For identification of purified proteins, we have access to state-of-the-art mass spectrometry instruments. In order to pinpoint specific interactors, non-specific proteins are filtered out using a multitude of methods, integrating quantitative MS tools and covering either a big dataset approach, control experiments or conservation of background across plant species.
More details on our protocols can be found in following manuscripts:
- Van Leene, J. et al. Capturing the phosphorylation and protein interaction landscape of the plant TOR kinase. Nat. Plants 5, 316-327 (2019).
- Van Leene, J. et al. An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes. Nat. Protoc. 10, 169-187 (2015).
- Van Leene, J. et al. Isolation of transcription factor complexes from Arabidopsis cell suspension cultures by tandem affinity purification. Methods Mol. Biol. 754, 195-218 (2011).
- Besbrugge, N. et al. GSyellow, a multifaceted tag for functional protein analysis in monocot and dicot plants. Plant Physiol. 177, 447-464 (2018).
- Nelissen, H. et al. Dynamic changes in ANGUSTIFOLIA3 complex composition reveal a growth regulatory mechanism in the maize leaf. Plant Cell 27, 1605-1619 (2015).
- Dedecker, M. et al. Transferring an optimized TAP-toolbox for the isolation of protein complexes to a portfolio of rice tissues. Plant Mol. Biol. 91, 341-354 (2016).
Selection of published stories including AP-MS data generated in our research group:
- Houbaert, A. et al. POLAR-guided signalling complex assembly and localization drive asymmetric cell division. Nature 563, 574-578 (2018).
- Heyman, J. et al. The heterodimeric transcription factor complex ERF115-PAT1 grants regeneration competence. Nat. Plants 2, 16165 (2016).
- Gadeyne, A. et al. The TPLATE adaptor complex drives clathrin-mediated endocytosis in plants. Cell 156, 691-704 (2014).
- Pauwels, L. et al. NINJA connects the co-repressor TOPLESS to jasmonate signalling. Nature 464, 788-791 (2010).