I’ve been working recently on purifying some proteins for use in future experiments and wanted to explain a little bit about what I need each construct for as they’re all for different things. Purification, as always, is a time consuming business but vital for any biophysical experiment that I do given all my work is in vitro rather than in a cellular context. It’s important to know what you’re putting in any given experiment so that you can interpret the results accurately!
The three proteins I was purifying were:
ALK2 – a short version of the construct containing the Kinase domain only, no GS loop, and a Q207D mutation that makes it consituativly active. We use this construct regularly for co-crystallisation studies and in this case I need some of it for growing some crystals for some mini-fragment screening later on.
ALK2 GS loop mutant – as described previously, we’ve mutated out various different phosphorylation sites in different combinations within the GS loop of ALK2 to see how this effects the activity. Overall we have 12 different version of this protein, this is one of them. I want to purify several so that I can compare them directly in an experiment all at once.
SUFU – so this is a slightly different project harking back to some stuff I did a few years ago so bear with me. In the BMP signalling pathway there is a protein called Endofin (ZFYVE16) which is involved in endosomal signalling/trafficking and may have some role in membrane localisation of the receptors and/or their substrates. In the TGFβ pathway the equivalent protein is called SARA and I managed to crystallise a previously unknown domain (a DUF domain or ‘domain of unknown function’) which is in the pdb as 4BKW. This domain had three sub-folds, two of which were known and one of which was novel. The two familiar folds looked a lot like SUFU which is part of the Hedgehog signalling pathway although there is practically no sequence similarity. It is known that Endofin and SARA bind to the protein TOM1 which is also thought to be involved in trafficking. To confirm whether there is a structural or sequence basis to the binding, one test would be to see if TOM1 also associates with SUFU – given the sequence differences between SUFU and SARA it is highly unlikely that TOM1 will bind but it’s worth testing just in case. I obtained a plasmid for an E.coli expressible version of SUFU from Christian Seibold and a large scale culture was grown up by Charlotte Manning, our new lab assistant.
I’m happy to say that all constructs expressed well and are now safely frozen at -80°C (GS loop AKL2 mutant and SUFU) or used to set up in crystal plates for mini-fragment screening which I’ll do a separate post about soon.
I’ve also manged to grow up a large scale culture of TOM1 (6L of cells) which I’ll set about purifying probably next week so I can then test whether it binds to SUFU.
You can find all the experimental details over on Zenodo.