More protein purification and some fine screens

As mentioned in my last post, I got 26 mg of ACVR1 on my last purification (as shown in my Zenodo log here). This is fabulous as it has allowed me to follow up a number of conditions for crystallisation. In particular, one of these hits was in G10 of my composite screen. I’ve therefore Read More …

Lots of protein now! A massive composite screen and a few crystal hits

Now that our workhorse ACVR1 construct is expressing again, I’m getting loads of protein – 26 mg on the last 3 L scale up! Because of this, I’ve been really busy in the lab trying to get things back up to speed again. While waiting for the ACVR1 to work, I purified some TGFBR1 (ALK5) Read More …

Virus titration and test expression of ACVR1 – some good news!

As I’ve mentioned in recent posts, our workhorse ACVR1 constructs have, for mysterious reasons, stopped expressing. This is clearly a massive problem, as with no protein we can not progress the project, and in the meantime, another DIPG patient has died, Aubreigh Nicholas, the 11-year-old girl who started the lemon face challenge. This is tragic, Read More …

Short form TGFBR1 (ALK5) purification and co-crystallisation with M4K2006 and M4K2089

In the absence of our “workhorse” protein, ACVR1 residues 208-499 (constitutively active Q207D mutant) (as we are still trying to work out why it won’t express at the moment), and the complete lack of crystals/diffracting crystals for the TGFBR1/FKBP12 complex, I have shifted to some other constructs we have to keep things moving. I had Read More …

Still no protein, and no diffraction from TGFBR1/FKBP12 crystals, but outreach is fun!

I’m sad to report that a couple of test expressions later, we’re still no wiser as to why ACVR1 isn’t expressing. It’s worked on a couple of small scale expression test plates, but just doesn’t want to scale up anymore. We may be looking at having to remake the virus. In the meantime, I had Read More …

Things still not going according to plan …

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 Read More …

A bad day in the lab – science never goes as expected!

I promised to post again today with an update on in-house screening of my hit for fragment screening, but sadly, there’s nothing to update, as yesterday turned into a litany of disappointment (not to be too much of a drama queen!). First, I started a fresh purification of ACVR1 to prepare for setting up new Read More …

Various purifications, and a few crystal hits

Gosh, it’s been a while since I posted – not because I have nothing to report, but because I’ve been super busy in the lab trying to get things done, as well as having loads of meetings and a symposium. So, in the meantime, I’ve done two purifications of ACVR1 and set up crystal plates, Read More …

The power of hope and community action: FOP family and friends conference – 19 May 2018

After the disappointment of looking through ~13000 crystal drop images over the past couple of weeks and finding pretty much nothing in most of them and nothing that actually diffracted nicely in the rest, it was incredibly heartwarming and hugely inspiring to attend the FOP family and friends conference in Manchester last weekend. FOP Friends is Read More …

Purification and co-crystallisation of TGFBR1 (ALK5) and FKBP12 with five ATP-competitive ALK2 inhibitors – so much disappointment

As you are probably aware by now, if you’ve read a few of my blogs, we are testing ATP competitive inhibitors to inhibit ALK2 but not any of the other 6 ALK family members. Thus, most of our activity and cellular assays test ALK2 as well as ALK5 activity in order to check the specificity Read More …