 Well, welcome to this presentation then. So I'm going to talk about production of B-cell 2 proteins involved in apoptosis and in our lab we are interested in apoptosis in general, but in particular the B-cell 2 family of proteins that I'm going to talk about today. So I'm going to directly dive into the apoptotic pathways. I'm sure you're quite familiar with this map. I mean it's a quite complicated process, but it has to be complicated because this is the ultimate control and checkpoint for removal of cells. So we can differentiate between the extrinsic and the intrinsic pathway. So when the cell is doomed to die, that signal is going outside the membrane to form the disc complex and then follow up by activation of a lot of effector proteins such as pro-Caspace-8 is getting converted into Caspace-8. Bid is converted into t-bid, but the ultimate termination of the cell is taken care about with the Caspace-3 and 7 that will fragment DNA. But on the mitochondrial level we have a tight balance between B-cell 2, the protein I'm going to talk about today, which is a membrane protein, and also BACs. So you can say that these have opposite effects, as B-cell 2 will inhibit apoptosis, whereas BACs will drive apoptosis forward. So but eventually when the cell is doomed to die, BACs will create the pore in the membrane and you get the release of cytochrome C that will drive this apoptotic machinery forward. So you can guess that if this balance between these two proteins goes wrong, and this is of course a hot topic for cancer research because it's been observed that cancer patients that have an elevated and even balance of these proteins. So the protein B-cell 2 is actually a family of proteins, they are main alpha helical, and also they have conserved BH domains which are important for the interaction between targets such as BACs. So as you can see here, B-cell 2, the protein of today's talk has a transmembrane part which is hydrophobic, so it's a membrane protein. It has four BH domains. I would say BH3 is the most important since it binds to BACs or other protein of interest. And we also have example of BACs that only has three BH domains, and you also have some old examples that even lack this transmembrane part. So the question we want to ask in our lab is basically a few key points here. So first we are very interested in pinpoint the location of B-cell 2 when it interacts with BACs at the mitochondrial level. And one thing that's turned to be the holy grail of our research is that we are lacking the structure of B-cell 2, which is an important protein to study of course. So this is something we are investigating and trying to solve the full length B-cell 2 structure. And also this means that there are no complex structure between B-cell 2 and BACs present either. But you can see B-cell 2 is a rather small protein, but it's a membrane protein, so it still has its tricks to purify which I'm going to talk about. So to the left here we have an NMR structure of B-cell XL. And I want to point out that this is not B-cell 2, but this is a valuable mimic of B-cell 2 because it's soluble. So what people in general doing, they are shopping off the c-termal hydrophobic anchor of B-cell 2 in order to keep it soluble. And they also have modified and removed some loop regions. And thereby you can make actually this molecule soluble, but however it's not the full representation of the intact B-cell 2. So our goal of our research is to work with a full length B-cell 2. And of course when you work with biophysics in general you need a lot of protein. And especially when you turn into NMR you need milligram amounts. You need a steady supply of protein. And when I started in the lab a few years ago we were relying up on cell-free expression method. And honestly it worked pretty nice. But the drawback it's a quite fiddly process. And we got maximum I think we got like a half milligram, one milligram of each batch. And it's quite time-consuming. It's not very easy to scale up. So I thought maybe you can do this with a recombinant expression system. And now I would show you this slide, but keep in mind that this slide is, I mean it's based on several years of works. I know it has been optimized and went through a lot of iterations. But I hope this might serve as a generic protocol for, I mean if you're working something similar maybe you can use this as a as a guide at least to help you out with if you have some expression problems. So what we're doing we are overexpressed. I mean this is like a standard recombinant expression at least in the first stage. So we overexpressed the protein in the pet vector, pet 15b, that carries some entermal histag. And we grow the cells at 37 degrees. And I even grow them overnight at 37. And the reason I'm using M9 media here is simply because it seemed to help with the overall yield when the cell grows a little bit slower. And also you can mention in LB the yield is not very great. And then next day I follow up with the resuspension in a fairly neutral pH buffer. And this step is it's only to create the inclusion bodies. So in this step we are not recovering the protein. Instead this recovering step is done in the next stage which is in presence of urea. So we are unfolding the protein and we add 0.6 percent of sarcosyl or sodium sarcosynate. And this seems to do the trick. So this sarcosyl it helps to resolubilize the protein. And then we run dialysis overnight against the detergent. It in our case happened to be pre-35. The reason why we use pre-35 is simply because it's cheap and it's available. And you can imagine when you do dialysis you need quite a lot of buffer and it would be quite cost-effective if you were using something else. And then we just continue with fairly standard affinity purification. We run a nickel column just on the bench. And we do a detergent exchange here into TPC. The reason why we use TPC is also that it seems to be very compatible with NMR. So we screen a lot of different detergents and this dodecylphosphoridicoline seems to be the best candidate. And then we remove the histag basically standard procedure with trombine and then we run the infiltration. And I just want to show you some examples of gel cell from each step. So first we have the iMac step. I mean you can see it comes off more or less pure immediately. I mean keep in mind this has been optimized quite a lot. Next stage we separate the histag and the pool here is basically the merged fractions from the previous step. And then we run the infiltration. And the good thing about the M9 we can now adopt in this procedure to accommodate carbon-13 or 1915 label. We can even do deuteration of the protein but then of course the gel will drop. But on average we get around three milligrams per liter of cell culture which is great for membrane protein. So something we also did initially was to compare the 4uV seed spectrum with the B-cell tube we obtained from this cell-free expression method. And it matches also very well with the recommended expressed B-cell tube. And just keep in mind that the x-axis is slightly different here. But we have confirmed that the fault is unaffected. So I was already talking about the DPC. So here is just an example when the full-length B-cell tube is run with NMR. So I'm quite sure you're all familiar with NMR so I don't have to dig into details. But you get correlation between the imide and the nitriene. And each black dot here represents one amino acid. And this slide will just show you that we can obtain a fairly nice HSQC with the full-length B-cell tube in DPC. And we can also apply this T2 relaxation filter where we can pinpoint more flexible residues in the protein. So how this works is that the flexible residues that we have a longer relaxation rate and then you can set the cut off for the relaxation and you can fish these out. And these apparently must come from the membrane of the micellar core region. Something we also are working on quite heavily now is, I mean I already hinted that we want to solve the structure of the D-cell tube protein. But in order to do that we have to deal with the fact that this is a quite large protein when you consider the added molecular weight in the micelle. So we have a problem with overlap in the spectrum as you can see here. But we still managed to assign basically all the loop regions. So we need to find a way to overcome this crowd and a more spectrum. And to do this we have developed basically a strategy where we can cut off the protein in several parts which is probably not a very uncommon strategy. But the good thing here which I'm going to show is that we can cut these and they will still work as small puzzle pieces to get the full B-cell tube spectrum. So you can basically overlay these smaller fragments that will form the full length spectrum. So this means we have truncated the protein, we have deleted this transmembrane part and we also modified the entermal sections of the protein in a quite careful manner. We have taken account that we don't interfere with the secondary structure of course. So again we have checked this with 4uV. And to the top left here you have the full length B-cell tube spectrum. And this is alpha helical protein so you expect a dip at 222 and 208 nanometers which we have of course. And then you can see to the top middle here the B-cell tube delta TM where we have deleted the transmembrane region. This also yielded a partially soluble protein but we still retained the helical fold. And the same holds true for the rest of these mutations. You can for as an example bottom left here we have deleted the entermal first 82 residues. We still have a folded protein. The same holds true if we in addition delete the transmembrane part. And then we have chopped off quite a lot of the protein but we still have the helical shape. Maybe one example in the end here when we only have the BH4 domain and the flexible linker domain. Then we start to see a spectrum that represents both a random coil and a helical spectrum but that's not unexpected for this construct. So just to give you an example of how this looks when you put it in the NMR spectrometer. So you can take an example here either A or B but just to show you that if you have the B-cell tube therefore the full length in black here we can basically superimpose the red and the gray and reform pieces of the full length B-cell tube which is nice and that also proves that we can use this method as a tool to overcome this overlapping problem. And also we have done some efforts in the reconstitution of B-cell tube into DMPC vesicles which was the first attempt. So how this works you have your protein in detergent and then you add lipid in our case we use DMPC in the first try. You form mixed micelles and then you reconstitute and you remove this detergent with biobeats as a quite straightforward process at least when something works it's straightforward but it worked so after two days you get you start from a perfect transparent solution you end up with a cloudy solution and then you form your proton hyposomes and then you ultra centrifuge and this is just an example how it looks like this is the real sample in the centrifuge tube and you can see that the B-cell tube and DMPC proton hyposomes has been trapped between the 15 percent and the 30 percent layer here. And then STSGL further confirmed that we can do this for even for this delta TM when the transmembrane part has been deleted we can also do it in the PUPC and corylipid which better mimics the mitochondrion membrane. So just to summarize a little bit so maybe the most important thing here we have found an expression method that works with B-cell 2 that is also a membrane protein and now we can yield milligrams amounts of protein and we have also incorporated B-cell 2 into DMPC and other lipid systems as well. We also found that DPC is the candidate when we want to turn to NMR it gives a very nice NMR spectrum and also in addition we can use these shorter B-cell 2 constructs to together to aid us to form the full picture of how the B-cell 2 protein looks like in NMR. So to finalize this talk I want to thank the group leader which is Gary Grebner who is probably listening right now and the rest of the group Amik who is doing some mostly of the NMR work nowadays and also some previous members of the group and we have collaborators Gothenburg and Prague and also in Lund and together with ESS and Neutron experiments are going on in Harwell. And finally I want to thank you for your attention. Thank you so much Jörgen that was a very interesting talk and having the aspect of NMR as well that's interesting. Yeah this was actually a 30 minute talk that I had to squeeze into 15 but yeah but it's so we are again a little bit late but just one quick question. Jörgen you're here to unfold your membrane protein so we are commonly using Jörgen to wash of course a little bit lower concentration than you're using but commonly membrane proteins do not really always unfold so to speak so you have STS present. Yeah I can give some input on that the reason I mean when we started to establish this protocol I was checking several papers and most of them actually used urea and then also which I don't show here we have tried without urea it actually works without the urea so it doesn't necessarily you don't need to unfold the protein to recover it but I don't think it's hard so we just kept on going with urea but you're right actually you may not unfold it that was my question. Yeah I think you will unfold it but I mean honestly we haven't tested that to run a CD spectrum in presence of have a concentration of urea maybe you can form some residual I mean secondary structures in presence of high concentration of urea in even in that system but I wouldn't be too surprised if it's not entirely going to unfold that that is probably true.