I ended last time saying Shubhash had saved the day by finding a better virus stock and growing up some more cells for me. Sadly, this did not work – still no expression. So we tried halving the amount of virus used, as using too much can infect too many cells leaving not enough making protein or new cells. Unfortunately, this did not work either – still no expression. This is very weird, as the expression of this construct is usually so good we use it as a positive control on test expression plates. So at the moment, we have two options: one is wait for the current test expression plate to be processed and if it works as positive control to scale that one up; or to remake the virus from the DH10bac cells (which could take two weeks).
Either way, I won’t have protein for a good few weeks. This is bad. No protein, no crystals, no structures.
In the meantime, I went to the synchrotron on Monday night to screen some ACVR1 co-crystals with M4K compounds (M4K2009 and M4K2045, and a couple with M4K3003 – check my previous Zenodo records for structures). While there was diffraction (apart from the few beautiful butterfly salt crystals I put in loops hoping they might have protein in them!), none of the crystals diffracted better than 4-6 Å, even the very pretty M4K2045 crystals. So. Back to the drawing board. We still have lots of compounds for screening of course, but no protein right now. Apart from that, I have grown no crystals of TGFBR1 (ALK5) in complex with FKBP12 and 11 different compounds (those with higher affinity for ALK5) in a total of 56 plates (with 16000 wells). It may be time to look at a shorter construct of TGFBR1 so that FKBP12 is not needed – although the complex seems perfectly stable enough.
Thus, the mysteries of science continue to perplex …
2 Replies to “Things still not going according to plan …”
Hi Ros! Sorry to hear about your problems with protein expression. The crystals sound promising, do you have more of them? I’m sure you’re already on it but perhaps you could try dehydrating them or seeding them in a new plate screen? Has worked for me in the past. Best of luck! Rachel
Hi Rachel, thanks for your comments. Expression still a problem, but I have some other mutants in the freezer I can try. I’ve put several of these compounds through seeding, but haven’t tried dehydration yet – next on list 🙂