 Rhaid i fewn gweithio. Rhaid i fewn gweithio'r yng Nghymru ar y cyffredinol i'n gweithio'r ffordd ychydig o'r bydd yma'n ymddangos y ffordd a'r gweithio'r sgwrdd ffins, gyda'r hyffordd ar y gweithio'r rhai cyffredinol yma'n gweithio'r gweithio'r ffordd. Can I just check, my screen is sharing it okay? Thank you. Rhaid i fewn gweithio'r ffordd, mae'n gweithio'r system wedi Allus, yma yw'r newydd pancell-baf, y syrthyn sythysydd. Ond ydych chi'n gwybod i Alas, rydyn ni'n gwybod i ddweud o'r gweithio am y maen nhw a'r maen nhw'n gweithio ar gyfer y Lav. Rydyn ni'n gweithio yng Nghymru, yma, yn y Nôl Fist yng Nghymru, ac yn ymddangos i'r ddweud i'r ddweud yn gweithio, ond mae'n gweithio am ysgol yma. Mae'r ddweud. A rydych chi'n gweithio'r ffamil amser o'r cyfwyr sydd ymgyrch i'r cyfrifiadau sydd ymgyrch. Rwy'n cymdeithasol i'r gymhreid y Debyg Llywodraeth yn y gwirionedd, i'r profesi Ion Hitchcock. Ion yw'r cyffredinol heimatologist ac mae'r lab wedi'i gweithio'r mylachol yng Nghymru o'r proses yw'r cyfrifio'r pethau o'r cyfrifio'r cyfrifio. A dyna'r rhan oherwydd mae'n ardal iaith yng nghymru o gwaith bod yna'r gwaith ym rydyn, oherwydd o'r cyfrifio'r cyfrifio sy'n dweud. Yr flyneddau rherwydd o'r proses yw'r cyfrifio, rhai o Rhymon, roedd yn Pwysig, o'n Tipo, ac yn ymrysau rhai o'r Chyfrifio, o'r Cyfrifio, oherwydd o'r Chyfrifio. NPL does not have any intrinsic kinase activity, so there's a third player in this game, which is Janus kinase 2 or Jack 2, which binds to the intracellular tail of NPL and transduces downstream signalling. So recently it's been shown by our group in collaboration with Jacopela Zab in Germany and also by other groups around the world that in the resting state in the absence of cytokine NPL and it's bound Jack 2 exist as a monomer at the plasma membrane. And then as the cytokine as thrombopoetin is enters the system, it's produced in the liver distant from its site of action in the bone marrow, where these receptors reside. The thrombopoetin binds to receptor, it has two binding sites and it brings two molecules of receptor together into this bridged ternary complex and this dimerisation on the outside of the cell translates across the membrane to bring the two molecules of kinase on the inside of the cell together into sufficient proximity that you get cross phosphorylation both of the kinases themselves and of the receptor tails. And this recruits other proteins to the receptor tails for phosphorylation and that's what triggers signalling. There are a number of ways in which this process can go wrong and cause disease and understanding these diseases is another aspect of our lab's work. There are diseases that caused by loss of function mutations which are distributed throughout the receptor. Many of these in fact I think pretty much all of them the mechanism of action if you like is that less receptor gets to the cell surface and when you have a lower receptor density, you get less formation of the active complex and less downstream signalling. And this results in reduced numbers of platelets and condition called thrombocytopenia. It's often a hereditary condition and this is very often fatal if untreated at a very young age. Some of these mutations act by interrupting with the folding and secretion of the transport of the receptor, trafficking of the receptor to the cell surface. Others the mechanism is less clear. Equally there are a bunch of gain of function mutations again distributed throughout the receptor sequence and these work by a mechanism generally of constitutive dimerisation in the absence of ligand. So even in the absence of cytokine you get receptor chains coming together in the membrane bringing together the jack to and downstream signalling. And here we end up with a raised platelet count or increased turnover increase proliferation of the hematorhitic stem cells in the bone marrow. Resulting in chronic blood cancers the second of which this primary myelofibrosis is almost invariably fatal. We know again through work done with our collaborators using live cell microscopy that these mutations in the transmembrane and juxtamembrane region work by stabilising the dimer receptor dimer in the membrane. And although we know that that's how it works we don't know at a molecular level how that stabilisation occurs yet. And the other mutations the by the mechanisms by which they work is relatively unclear. So you'll see that I've drawn this receptor as a schematic cartoon because there are no structures available for it in the public domain at the moment. And there are several reasons for that. It's a relatively medium sized protein I suppose compared to what we've been learning about today. It's a bit too large and too flexible for x-ray crystallography. It's a bit too small for cryoEM. So what we do know from sequence is that the extracellular domain is comprised of two modules called cytokine recognition modules. The cytokine binds to the distal, one of these the further away from the membrane and then there's a proximal domain. Each of these is further subdivided into two subdomains, one of which is likely to be immunoglobulin like fold and the second a fibronectin like fold. The immunoglobulin like fold is held together by two disulfide bridges and then each of the cytokine recognition modules is like oscillated in two places. So it's quite a complex system, not perhaps as complex as some of those we've heard about today, but it has its own challenges. Then there's a single transmembrane helix and what is predicted to be an intrinsically disordered tail which probably folds upon binding to its various binding partners within the cell. So there's there's homology between this distal region that binds the cytokine and related receptors such as the erythropoetin receptor and that allows us to build a homology model for this distal region. We can assume that probably the next module has a similar structure, but the relative orientations of these two modules with respect to one another and to the membrane are completely unknown and maybe important for function. So on top of those characteristics of the protein itself, one of the other reasons that it's difficult to study and that there's no structure available at the moment is it's difficult to make. It's tightly very, very tightly regulated and physiological expression for good reason in that you don't want your the numbers of blood cells circulating to be dysregulated that causes disease. And the main cell or non cell on which it's expressed in the body platelets, there's less than two receptors per square micron, which equates to a round about less than 100 receptors per platelet. So we worked out that if we wanted to make it from natural sources, we'd need the platelets, the full platelets for the entire blood of about six different people or one cow. It might be possible to obtain this using afferized expired afferized platelets, which are kind of a concentrated form of the platelets from blood. And we did have a plan to do that, but that was disrupted reasons that many things have been disrupted over the last 12 months or so for everyone. The overexpression system that many labs use for doing functional studies on MPL, these B cell based mouse B cell cultures, even those express maybe a maximum of 5000 receptors per cell and again very large quantities would be needed. So I've had a go at making intact receptor expressing intact receptor in insect cells. This was very preliminary work. We could show by flow cytometry that a small number of the GFP positive so bacula virus infected cells did have receptor on the cell surface. And I was able to at least show expression in total cell mass by Western blood. But this is a very, very low level of expression and there's quite a lot of degradation. And I have no idea what the yield would be. So why go for a cell free synthesis system when I'm working in a wonderful department that under normal circumstances I have access to expertise facilities and equipment all across the department. And I could do this in almost any expression system that I wanted. Well, as I mentioned earlier, and as many of us know, over recent months, our movements have been somewhat restricted and we were asked for a long time up until very, very recently to basically concentrate on what we could do from our own benches. And for a while, all I had was my laptop at home. And, and what I have at my bench, I can do molecular biology, I can do very small scale purification using my magic multi pet system that has risen for the tips. I can do gels and a few other things, but I certainly didn't have access to the equipment to do large scale grows in P here or insect cells or anything like that. And while we were at home, I was contacted by this company Lenio bio, who were looking for early adopters for their cell free expression system. They've developed using plant cells. So I thought I'd give it a try. So who or rather what is Alice. So I quote, Alice is a scalable high efficiency eukaryotic selfie protein. And what this means is they the system is made from tobacco plant cells, which have been lies. And they contain intact organelles, including mitochondria so they can provide their own energy supply. And they contain microsomal structures that are then allow for the folding of complex proteins that need disulphides. And they also have all the mechanisms for doing your glycosylation, at least end link glycosylation. It will be slightly different glycosylation than in a human cell because it's a plant cell glycosylation, but at least it does some. So it sounded quite interesting. And also because of the microsomal membranes, it means you can consider looking at integral membrane proteins. It's really, really simple to use. It's supplied as 50 microlitre aliquots in two mil tubes. And essentially you take your aliquot out of the freezer, defrost it and add some RNAs free DNA. And then shake it for 24 to 48 hours in a room temperature. Try to keep the humidity. Constant. At the end of your reaction period, you add detergent. So in this case, I used half a percent or 1% dodes on multi-side. Salubalise and lyse the microzones and salubalise your protein of interest. Spin down, take off the supernatin, do some affinity chromatography. And if you want to do this in multi-parallel mode, then you can use a multi-parallel mode. Then you can use a plate-based setup as well, which is a, you can get away with 25 microlitres of material. So it was, it was very, very, very simple. You need to make your DNA in the right, to put it with the right surroundings, if you like, to enable the expression in the plant cell-based system. You can code on optimise if you want to, I didn't. I just took what I had in the freezer already in terms of, of gene. The plasmid that you can use with a system is that there's one for a cytoplasmic expression, or the equivalent thereof. I'm using the one that PLS2, which is for microzomal expression. So for membrane and secreted proteins. It has a T7 promoter. Then there's the five prime leader sequence of the tobacco motoric virus. This is followed by your, where you put your gene of interest. The vector contains a signal peptide, the melitin signal peptide, a strep tag, a factor 10a cleavage site, yellow fluorescent protein, a six histag, and then the three prime-line translated division. What I chose to do, which with hindsight and listening to all of the talks, with perhaps not the most sensible approach at the time, was to clone my MPL in between the signal peptide and the histag, because I knew from previous work that having a histag on the C-terminus was not likely to be functionally problematic. I didn't at the time have access to treptactin redin, but I did have access to iMac. I looked at both the full length protein and also the ectodomain construct, because I know this can be made in mammalian cells, and I was using it as a comparator. My very first expression test, if you like, if you can call it that, reaction test perhaps is a better word for it. I use the generic conditions which are used for cytosolic proteins, and here I'm showing gels for EYFP as a control compared to my full length and extracellular domain of MPL. I let the reaction carry on for 48 hours, and I used 5 nanomolar plasmid, and then I added detergent to the whole reaction and solubilised that. And I was blown away to be able to see, I hope you can also see where I've drawn these arrows, that there was a clear and quite strong band in my total samples, or both the full length receptor and the ectodomain. When you go in with an antibody in the western blot, the optimism goes away a little bit, I guess, because there's also an awful lot of smaller truncated products, and when I look at what's in the soluble fraction, it's nothing like what there is in the total fraction, suggesting that some of this, or quite a lot of this, is not perhaps correctly folded. And that's the same for both of these constructs. So it turns out that particularly for challenging proteins and for membrane proteins, proteins expressed into the microzone in particular, less is very much more, and I think this is echoed by some of what Chris and other people have said over these last few days, that you need to tune your expression system so that it can cope with the processing and folding of the proteins that you're making. So I scale back to 24-hour reaction, after which point the microzones start to be less happy, and I halved my plasmid concentration, and those are very much, pretty much, that the main variables that you alter with this system, apart from, of course, your construct, as you might do with any other system, and this method I no longer, although I no longer see a strong band for the proteins of interest in the total sample, there is, I think, a band at about the right size for my extracellid domain in the eluted material from my iMac column, and when I look at my western blocks, it's not completely perfect, it's a neat, cleaned away, a lot of the truncated products, and I now have two bands, and I'll talk about where these two come from in a moment, which is, and there's more, certainly, for the ectodemane, much more of the upper band than there was before. I did a couple of other changes in this step, as well as reducing the reaction time and plasmid concentration. I introduced an extra step of microzonal isolation before I solubilised, and I used a bit more detergent in a solubilisation time, and all of this, I think, has helped to clean up what I get off the column, although there's still a lot of contaminants, and I'll come back to that in a bit. Uh-oh. There we go. So, yes, as I mentioned, I get a doublet for both the full-length protein and the extracellid domain, and my suspicion, or my hope, I guess, was that this was due to glycosylation, so I treated all my samples with In-Gaze F. To look for any linked glycosylation, which I expect on this protein, and on the treatment with In-Gaze, I see my double band collapse to a single band at lower molecular weight. And interestingly, and another demonstration of less is more, as I increase my reaction time and increase my DNA concentration, I decrease the amount of glycosylated protein as I'm overloading the system, I guess, and it can't cope anymore. So, I've got glycosylated protein. At least some of it can be solubilised in detergent. And the next step, as we've heard over the last few days, will be to show that it's functional. And I've used a number of different methods to attempt to do this. First of all, I made, so we have plenty of the cytokine in the freezer, and it happens to have an N-terminal serine. And so I used a method that's been developed in the chemistry department for labelling, site-specific labelling, the N-terminus of a protein that has a serine at the N-terminus with an aldehyde-based probe. And in this case, I used a biotin probe. This method's called the OPAL method. Organo-catalyst. Now, I can't remember what the abbreviation is. Organo-catalyst protein aldol ligation. I think that's got it right. And this worked really well. It's got 99, sorry, 98% biotin elation. Then my thought was that I would bind MPL and TPO together and pull out the complex on streptavidin beads. Unfortunately, what I found when I did the pull-downs was that even in the absence of TPO, biotin elated or otherwise, the MPL bound to the beads anyway. So although I did get pull-down of my reaction products, I can't really say whether that's because or just because of nonspecific binding to the beads under these conditions. So that's something I need to optimise. The next thing I tried was exploiting the histag on the MPL to see if I could then pull down TPO onto my nickel beads, onto my iMac column. And again, I do see TPO in my eluted material, which was encouraging. However, when I look at TPO alone in the absence of MPL in the presence of 10 millimolar imidazol, which is what I used for this experiment, it's quite happily, even though it doesn't have a histag. I can reduce that nonspecific binding by increasing the imidazol concentration, but I haven't yet had an opportunity to check whether my MPL is still able to bind stably to the nickel resin at these sorts of levels of imidazol. Hopefully it will and I can reduce the nonspecific binding that way. The third method I've tried, the FAR, is using chemical crosslinking with this compound called BS3, which crosslinks lysine residues or free amines on the two proteins in your mixture and also on the proteins themselves if they happen to form oligimus. And unfortunately, again, that's what's happening in that, although I do get higher molecular species forming and visible by SDS page, in the presence of the crosslinker, I get those in the absence of binding partner as well as in the presence of binding partner. So, again, I can't really say whether or not the protein is functional. So, lots of things to try. Our department is able to open up again now, and so I'm hoping I can get access to a suitable analytical size exclusion system to find out whether the MPL that I've made in this system is monodisverse and what oligomeric state it has. There's lots of work to do on the pulldowns to try and reduce the nonspecific binding and optimize what's going on there. I'm hoping to get access to a fluorescent label that I can use with the OPOL method to label my ligand to enable further assays. We have access to micro-scale thermoferesis and a plate reader for thermal stability assays. The protein that I have made needs much more purification, so it turns out that a number of the proteins within the cell-free system are themselves hystagged, so using a hystag wasn't necessarily the best way to go, but because my protein is glycosylated, I'm going to go back to a lovely old method that was used for rhodopsin and see if I can get to enrich the glycosylated material on concanabalin cepheros. Then much longer term, I'd really like to try the system because you have so much control over what happens and what you put in. I'd like to use it for co-expressing MPL with TPO and JAK2 to make the full signalling complex. Then there are a whole bunch of other wonderful ideas that this workshop has given me to try. Thank you all for your contributions. In summary, I've used this new plant cell-based cell-free system to make full-length MPL and its extracellid domain. These are expressed into the microzones. I can solubilise them with detergent. They're glycosylated, at least at some extent, and I can purify them partially with the hexasidine tag. I think I'm in a bit optimistic here about the yields. I've done some recalculations. It's probably much less than a megamil, but that's still not bad compared to what we get in other expression systems. I'm still waiting to find out if it's functional. Watch this space. I'd just like to finish by thanking everybody past and present in the Hitchcock lab, particularly my PI Ian for giving me support and letting me just get on with things really, giving me a lot of freedom to try stuff out. It's a cell biology lab, and so we've had a good time finding out one another's vocabulary and language and then to speak the same language. I'd like to thank Tess for doing the flow cytometry on the insect cells, and Gian and Sophie for being able to get into the lab on days when I wasn't able to get in to do things like rescue plates and put on overnight. A lot of our work is funded by Cancer Research UK, so we're very thankful to them for funding. Obviously, a big thanks to the Folk at Linear by my neck, Ricardo, Frank and Joanne for their technical and other support, and then members of the biology and chemistry departments for technical support, reagents and their expertise, and Max in Osnabook, whose figures I pinched for some of the introductory slides. I'll just finish, if I may, with a bit of promotion in both the Hitchcock Lab and Linear Bio are currently recruiting, so if you'd like to apply or you know of anyone who'd like to apply their skills to experimental hematology or protein glycosylation, then please get in touch with Ian or my neck, respectively. Thank you, finally, or very much for listening for all your ideas and thoughts over the last two days, and I look forward to taking your questions. Thanks, Julie. That was a really wonderful talk. So we have some questions. Why is MPL difficult to collect with x-rays? So it's the flexibility as much as anything else, as well as the very low expression levels. It's very, very hard to make enough of it. I think you could envisage trying to do crystal structures of the two separate cytokine recognition modules if you were able to identify the junction between the two of them reliably. Now, what I haven't shown today because there wasn't time, I did a lot of screening early on in insect cells looking at portions of the extracellular domain, the distal and the proximal cytokine recognition modules and trying to identify boundaries between them. And the best expression that I got was for the full extracellular domain and trying to make smaller subunits generally ended up in this folded material that didn't get secreted. So it seems, although it looks from sequence, as if the two modules might be independent, I think actually structurally they need to work as a unit. Okay. So, the next question is, how about adding nanodesk, smalps or liposomes to others? I'd love to try that. I'd really love to try that. I don't know, I'm guessing because you probably want to keep the microsomes intact through the reaction process that you'd want to put those in at the end as an alternative to solubilisation. But I definitely want to give that a go. So, again, I'll let you know. Yeah. So, another question is, why Alice Free's expression system? Do you run it in reducing conditions? And also, do you see current disilfined bone formation in the samples? So, as I say, I haven't been able to demonstrate functionality yet. So, I can't say for certain. And I don't have an assay that lets me look at whether or not the disilfined are there. However, within the microsomes, you should have an environment that allows for disilfined formation. So, any protein that's produced outside of the microsomal compartment, it's unlikely that it will be properly folded. But protein that's produced in the microsomal compartment should be formed. It's possible to make. So, I haven't done this, but there's quite a lot of literature on using the system to make folded antibodies and other proteins that require disilfined formation. And that seems to work really well. I don't have those data to share with you today. And as I say, that wasn't done by me, was it other eukaryotic self-resystems? I haven't. I don't know whether Leniobio have. I know they've surveyed the literature, but I don't know whether they've done an actual experimental head-to-head. So, I could take that question to them and get back to you. Okay, so thank you Alice for this amazing talk.