Protein Production Sweden with Malin Bäckström

Hey, I think we slowly start. So, it's my pleasure to introduce now, I think, the fourth webinar that we have in the Panda theme series. And today it's about enabling technologies. So, how can we get help to prepare the samples that we need for the experiments? And today's speaker will be Malin Backstrom, who's from Gothenburg University, and who is also the uh director of the national infrastructure for protein production here in Sweden. So, as I understand, Malin received a degree in biochemistry and then did her PhD in medical microbiology. I guess everything in Gothenburg. But then also spent some time in Cambridge. And for many years then worked with Musse in uh biology at the University of Gothenburg. But then you focused very much on service and core facility. And from 2015, I think, if not even earlier, you're driving and leading the platform at Gothenburg University that is doing especially expression in mammalian cells. But then since 2022, we also have a national infrastructure which you are heading. You are the director. And that's what you will tell us about today. And in particular, as we talked a bit before, a few examples also what you produced for virologists and virology projects. So, with having said that, please, Malin, go ahead. >> Mhm, thank you, Wolfgang. Uh I will then share my presentation. Yeah, so thanks a lot for this introduction and thank you also for allowing me to speak here today to this group of people. Uh as Wolfgang said, I'm heading the the national infrastructure protein production Sweden, uh which has the purpose of supplying all researchers with the proteins they need for many different types of research projects. Uh and of course virology is a hot topic and we believe that many of you also will need proteins for different things. So it's a very good idea that Wolfgang had that we should be should be allowed to present PPS here and tell you what we can help you with in relation to to protein production. Uh so I will present PPS of course today and I will also present a little bit of my own uh platform core facility at the Sahlgrenska Sahlgrenska Academy in Gothenburg. Uh and we are working with we are we are part of this national thing with PPS, but we are working with mammalian protein production. And at the end I will give you two examples of projects that we have made that are related to to different virus projects. Okay. So um Uh to first then I will introduce this protein production Sweden for you who have maybe not heard about us before. We are a national distributed research infrastructure for protein production and purification. We are national, we have national funding from the research council, but we are distributed because we are located not only in one site, but in actually in five different universities in Sweden. So we work with open access for projects from both academia and industry, both from Sweden, but also from other countries. And we what we found out, I mean, when we established PPS, we we found out that we had protein expression platforms at the different universities in Sweden, but that we were working with different expression systems and we thought that by working together we can help all researchers, I mean, in a combined way. So this is why we wanted to set it up. So we have a a one one single infrastructure that the researchers can turn to and then get help with production of proteins in the different expression systems. And as I said, we have funding from the research council and of course also then from the participating universities. And this was established in 2022 and at present we have funding until 2030 because we just recently got the full extension of of our funding. So what are we? We are several different protein production platforms in four different geographical nodes, but at five different universities. So if we start here in Gothenburg, where I am, we have my unit here, which is the mammalian protein expression core facility. And we are located at the Sahlgrenska Academy in Gothenburg. And also in Gothenburg we one platform which is the Swedish NMR center, then they make then cell-free expression of proteins. And also can do a lot of labeling for NMR, but also for other things, of course. And in Gothenburg, there is also one platform working with Pichia pastoris, called the yeast protein production platform. So, they they are the three labs that we have here in Gothenburg. And then in Lund, we have Wolfgang's platform, the protein production platform LP3 who are working with within PPS, they're working with insect cell production. And then we have two labs in Stockholm, one at KTH and they are working with mammalian production also, as we do here in Gothenburg. So, we have two platforms for mammalian cells within PPS. And then we have the protein science facility at the Karolinska Institute, and they are working with E. coli since many years. And this is what they contribute with in this in this context. And then we have Umeå University who who have their protein expertise platform working with E. coli and also establishing a bit of plant cell expression. I will come back to that later. So, this is all the labs. So, we have two labs E. coli, two labs working with mammalian cells, and then the other ones, one for insect cells, one for yeast, and one for cell-free production. So, this is what PPS is. And I don't know now who who's actually listening to this webinar, how much experience you have in protein production. But this slide shows that there are many different steps involved in getting uh uh good protein of high quality that you can use for different research experiments. So, first of course, uh you need to um to uh look at the the gene. I mean, the protein that you want to have expressed, you have to look into the the gene for that and make the design and see how should the protein what should be included, excluded, etc. And then of course, you have to put it into the right expression vector so that you can express it express it in the in the expression system that you're then then choosing uh for your specific protein. Because the reason why we have so many expression systems is that there are so many different types of proteins and they will have different properties that will make them well, to be better produced in in one or the other expression system. So, this is why we want to work together and have complementary systems so that we can be able to produce many different types of proteins. So, when we have designed the vector, uh decided how the protein should be made, in what cells, etc., what expression system, then we make the actual production. And that could be a large scale culture or it could be in when it comes to cell-free, it could just be in a test tube. But the the the actual production step. And after the production, we also make purification of the protein and that could be either from the cell supernatant or from the broken up cell pellets depending again on how you design your experiments. And then we performs uh uh column purifications. And at the end we also perform some quality analysis because we have to make sure that the proteins that we deliver from PPS are really of good quality. So we have [clears throat] also included that in our workflow. We have a vision in PPS that we shall become and remain the preferred provider of proteins for research purposes. And that could be for Swedish Swedish researchers from academia, public sector and commercial entities. So we have now worked together for almost 5 years and uh uh we have started to work uh and established our workflow that we so that we can work together. But we also uh as we say here, we also want to remain the preferred provider. So we still always have to keep up the good work and make make proteins of good quality so that people want to continue uh using us. Uh we are of course aware that many labs can also do recombinant protein production in in their own lab. It happens quite a lot in many labs. Uh but we see that we want to help also the ones who cannot do it themselves. Or it can also be a time-saving activity for even for the labs who can do it themselves. They can even lease it out to us if they and then they can focus on the the other steps in the research project. So we're really um Yeah, we want to I mean we we working to get more and more known. It takes time of course to get known and we want people to to to use us and want to continue to use us also. So this is a slide of why of our workflow that we have in PPS. Uh as I said, we want to work together. So we have one uh common access point on our website. Um so there is a web uh form that you can fill in for a project request. And then when these projects come in, we see all these. We have them they come into our database and we look at that in our in the management group. And the management group consists of all the heads of the different labs. So then we can discuss, "Okay, we this researcher want to have this protein. What is it? Who can help? Where should this project go?" And then we distribute uh the projects to the different platforms. Uh and as I said, it depends very much on the the proteins. The the properties of the actual protein where uh which platform the project should go to. And here again are listed all the different expression systems that we are working with. We also have two uh so-called gateway uh modules. Uh they are both located in Lund and these are then very much aimed for um for uh production of proteins with uh deuteration. That's directed towards the ESS. And we also have one gateway module called molecular chaperones and these are proteins that could then be uh intended for for example for co-crystallization. Uh so antibodies and different fragments, etc. So they are kind of specialty uh modules. So and then the work is done in the four different nodes. And at the end we also perform, as I said, quality control of our proteins. We all all of us do that and then we deliver to our users. So we have six expression systems, two gateway modules, and we perform purification and quality control at each of the nodes. Now, when it comes to a quality control, quality assessment of the proteins, we have agreed uh between us that we want to focus on three important things that are really crucial for a protein to be of good quality. Uh the first one is purity. Uh and purity can be assessed by a Coomassie stained gels. This is quite standard and quite simple, but in a gel you can see uh how your protein looks and how pure it is. So, that's a that's an important thing to check for. The second thing is identity of the protein. So, all the proteins that we make, uh we also send for mass spectrometric analysis to verify that the protein is actually what we believe it is. Uh there are several different ways that we can can do that. Uh either you can run a protein in in intact mass, I mean the whole protein to get the mass of the whole protein. You can do that with smaller proteins at least. Or when it comes to larger proteins, it's better to do tryptic digests and then analyze the the peptides. And I'll show you one example of that here where we uh all the the green peptides here were identified in a mass spectrometry experiment. So, we could see that this is clearly the protein that we intended to produce that we have here. So, we verified the identity. The third thing, which is also very important, which is sometimes a little bit forgotten maybe, uh that you have to check that the protein is the right conformation and that it is a preparation of mono dispersity so that you don't have a lot of different molecular forms or you have aggregated proteins. You really want to see a nice sharp single peak in an experiment like this. So that you don't have several different peaks that could that could indicate that you have some maybe aggregation or so. So we always aim for that. And this can be checked by size exclusion chromatography or other analysis that also involves separation by by size. And we have said that either we I mean we often have SEC size exclusion chromatography as a second purification step and then we know that we have only pulled the fractions so that then gives this sharp peak. But if we for some reason don't have that purification step, we also we say that then we need to do this analysis in another way. So all these three aspects are of course important to know that the protein is of good quality. Okay and then this slide shows that the proteins that we have produced and delivered, they are used for many different areas. I think many people maybe are thinking about structural biology when you think about recombinant proteins and protein purification. We see of course that many of our users do that. Crystallization but maybe nowadays more even more common to use cryo EM. And some part of our proteins of course go into structural biology but definitely not the most anymore. So they are used for many other things as well. They are used for different types of biophysical and biochemical characterization, different methods. They can be uh used for drug or vaccine development or to develop antibodies or check the antibody the binding of antibodies, etc. Uh then for the development of different uh technologies or methods, uh ELISA, for example, we produce uh quite a few antigens for for uh for ELISA diagnostics, for example. And then the proteins are also used for assay development and screening or in cell cellular experiments. We don't produce anything for clinical use, so it it's uh only for for research use, the proteins that we produce. But there is a a big variation in uh in the use of our proteins. Okay, some numbers. Uh these are numbers from the first 4 years where PPS existed and worked together. So to the right here, we have the total number of projects and PIs. Uh this is then from the old labs combined. So you see the applied project for each of the four first years years here. So you see applied projects, completed projects, and then the number of researchers, PIs, with applied projects and finished projects. And the last bars here are for delivered batches. How many protein batches that we actually delivered from the different labs in total. Uh and if you sum this up for the different years, uh we have produced more than 1,400 proteins so far uh from PPS. Uh I should mention then that around 50% of that is produced in E. coli. Uh E. coli is uh uh still quite a popular system and uh they are also quite fast. So they can produce more proteins that several of the other platforms can. But again, it depends on the nature of the protein whether it's possible at all to produce in E. coli. Not everything will be. So there again, it's nice that we have these complementing systems so that we can help more researchers with their proteins. And down here to the left I put in the numbers for project applications from companies and from from abroad from other universities not in Sweden. And we we also see an increase of the projects coming from these players which is of course very nice. So we see a nice development and we know that we have helped many researchers so far and as I said we try to keep this up. Keep this going. Also for the future. And the way to get access to our services you make a project request on our webpage. And here are some you can find on the webpage details of what it is you you need to enter into the form when you make a project request. So it's it's about the protein. And what you want help with and some other things. And of course this is not a binding. It's not that you have to do it have to make the production. So if you have if you're interested and you want to get more information or you want to discuss a project before you actually make kind of an order. This is the way to start because when we have got the project request we can also look into it. We can discuss it with you and see what will be feasible and what we want to do and then we will give you a quote or a price estimate for the work and then you will decide whether whether we should then go for the project or not. So sending in a project request it's not a binding thing. It's not that you have to do it afterwards but it's a it's a good starting point for for coming in contact with us so that we can discuss your project. Okay, so that was very general about what PPS is and what different expression systems we have within us. Now I have one slide for each of the expression systems. So this is slightly more detailed. And then I start here with the E. coli production which I already said that we have in two places both in Umeå and at the Karolinska. Uh So they work with some many different vectors and cell strains. They can perform small scale screening. They can make NMR or isotope labeling for example quite easily in E. coli. And this expression system is a comparatively cheap and fast. So if you have a small protein or a simple protein or I mean you can also make domains of larger proteins for example. So if you can if you can design your protein in that way it's a it's a good system to start with because it's it's quite cheap. But and fast. But of course it's not always possible to have your protein expressed in E. coli. If if the protein has several disulfides they can also work with periplasmic expression. So it it enable to to get the disulfide bonds formed in the correct way. So they have some tricks to do things in different ways. They also have set up the incorporation of non-natural amino acids, which could then be used for different labeling, etc. So this is also something that they can offer as a service to do that. And the next system is yeast. And the system they are working with, this is here in Gothenburg. They work with Pichia pastoris, which is a eukaryotic cell, but it's quite simple. It can make post-translational modifications, and it can fold proteins, complex proteins in a in a better way than E. coli. So this is a system that can be used if E. coli doesn't really work out. And yeast, Pichia, has the advantage that you it's quite inexpensive to grow, and you can get very high cell densities, and they're working with a very strong promoter. So you can have a lot of cells and get a lot of protein produced when you when you induce the production. They work with secreted proteins, but these cells are also suitable for a large variety of eukaryotic membrane proteins. So this is also a platform that can be used for for membrane proteins, which are more tricky in in general to make, but these cells could be a good starting point. Again, they can do isotope labeling for NMR in these cells. And uh the way they work is that they make if they get a request, they make a stable cell line or or clone before the product that produces the protein and then they can store that and use that again. So, they can make several batches without having to start from the beginning. So, that was a little bit about that. Oops. And then I turn to the insect cells uh which is then done in Lund by Wolfgang's group. Uh this is also eukaryotic system uh that is a little bit more Well, can work with more complex proteins than than normally uh E. coli. Uh you can also get a very I mean, here you make a virus uh baculovirus that you infect you use to infect the insect cells. Um so, it has some start uh it's start up as I cost and work to do the virus uh but then of course, you can produce the same thing again with the same virus. This is good for production of mammalian cytosolic proteins, not so much for secreted maybe, but like intracellular and different types of enzymes etc. This could be the system of choice. And the next system is the mammalian cells which is the more the most complex uh cells of course uh in that we have access to in PPS. Uh we We are two labs doing this at at KTH and here in Gothenburg. And we both work with the mainly CHO and HEK cells. These are hamster cells and human cells. So, we have both these cell lines going in our labs. So, we work primarily with transient transfections uh at least for making medium batches of protein when you don't need too much. If you need more, you may be able to to want to make stable cell lines or clones. But that's not so often needed any longer as if you don't need too much of protein. Uh we work with adherent or suspension cell cultures. We also have the ability to grow cells in bioreactors for larger batches. Uh and then grow the cells with perfusion. So we can grow cells in many different formats. Uh we also have the possibility to work with different cell lines uh that will make different patterns of glycosylation. Uh so if the glycosylation, if you have a glycoprotein and the glycosylation is important, then of course these are the cells to use and we are able to modify that in uh in different ways. And one thing here which may be important for some of you is that uh when you work with mammalian cells, you don't have any endotoxin contamination in the protein preparations. And that could of course be important when you're doing say immunological experiments or other biological experiments where where the endotoxins can make a can be a problem. So then this is a good system to use. Mm and then the Umeå lab, as I said before, they have also set up plant cell suspension cultures for protein productions. Uh so there are um this is not not the most common thing to do, but uh it's also one eukaryotic system then that you can use. Uh also again free from endotoxins. Um yeah. So, this is an alternative sometimes. Okay, and the last system is then cell-free. And this is done also here in Gothenburg at the NMR center. And this is then just made without cells, exactly as the name says. Uh you just mix in a tube the the plasmid DNA with the gene, and then you mix it with an E. coli lysate. You mix um you just wait. This is a very quick system. And again works for more simple uh proteins cuz it's E. coli based in a way. Uh but here also it's quite easy to check uh or or modify uh culture condition or not culture, but um reaction conditions like pH, temperature, buffer, redox conditions, etc. So, they have the possibility to do this in in parallel for trying out different uh conditions for the production of the protein. Okay, so those were the expression systems. And here again I will mention the gateway environments. Uh one is for making protein duration of protein uh for neutron molecular molecular crystallography or use at ESS. And the other one is then for to aid in crystallography crystallography as I said. Uh for example, making antibody fragments that you want to have your protein in complex with, etc. to aid for crystalli- crystallization. And these platforms are located in uh in Lund with Wolfgang. Okay, so that was a an overview of uh of PPS, what we can do. Uh I will tell you a little bit more now about my lab uh and the core facility that that we have here in Gothenburg for production in mammalian cells. Uh so uh we have been working as a core facility here at the Sahlgrenska Academy for for 20 years by now. And what we have focused on is the production of complex proteins and for example glyco proteins. And we can culture cells in many different formats uh both adherent and suspension cultures. Most of the time we use shake flasks, but we can also make bioreactor cultures for for even larger batches. Uh we use Chinese hamster ovary suspension cells. Uh we actually started out with that. When we started as a core facility, we were working very much with CHO cells. Uh this is also what is very much used in biopharmaceutical industry for production of of recombinant biopharmaceuticals. So, it's a it's a cell line which is um approved for for that type of use, also. Uh but over the years uh these HEK293 cells have become and more and more developed and they are very good at producing high amounts of proteins. So, so we are turning more and more to to these cells instead. But we still have the possibility of doing it in CHO cells if that is needed for any reason. It also happens sometimes that we get the request just to deliver cell uh uh cell mass. Not not cells producing specific proteins, but just to get a lot of cells of some kind. So, that we can also help with. Sometimes people want to have a lot of cell mass for different types of as a as a starting material for these different types of research experiments. So, we can also just grow up a lot of cells. So, here are some pictures from our labs. This is known probably to most of you quite common cell culture facility. What is maybe a bit more unusual is the use of these bioreactors that we have. We have 3 L vessels. Where we can also culture then suspension cells. Um And here is a slide showing a perfusion culture. What we do then is that we have a bioreactor with a stirrer. We have electrodes for checking pH, dissolved oxygen, and temperature continuously. And you keep control of these parameters to keep cells in a good conditions. And this way you can keep the cultures for longer time because we have better control and also then because we have this perfusion meaning that we have a cell um cell suspension here in the vessel in the middle in the actual bioreactor. And then we feed in medium. And we feed out used medium spent medium then containing the protein of interest especially if it was produced as a secreted protein. It will end up in the supernatant, and we can harvest continuously also. And then we have something called a spin filter in the middle here, so that we keep the cells in the reactor and only harvest the solution, the spent culture medium. And in this way we can get very high cell densities and in this also then a good production of the protein. And we can do this uh I mean, you can keep the cells for some time, maybe a couple of weeks, and harvest up to 20 L from a reactor like this. Okay, so that was a bit about the production. We're not of course uh not only doing it in bioreactors, we also make a lot of shake flasks, but then after that we also need some post-production processing. Uh first we make a concentration if that is needed, especially if we start with 20 L, it's better to make uh concentration first. But then we work a lot with these column chromatography, which we all of us do at the different platforms of PPS. We we use uh His-tag purification, size exclusion, ion exchange, and other things as well. So, this is I show you pictures now from our lab from my lab, but of course that is something that we do all of us. And then different types of analysis for to look for uh yield and purity of of the protein that we produced. And again, as I said, the the quality control. And then we deliver to the to the researcher. >> [clears throat] >> So, this slide shows uh one specific example where where where we generated one um it's a viral protein. It's actually a receptor-binding domain of the SARS-CoV-2 spike protein that we produced at uh our lab, and we were then for a research project, we wanted to produce the same protein but uh with different types of glycosylation. So, we tried different ways of modifying the glycosylation. >> [clears throat] >> So, we use some variants of CHO cells and also some variant of 293 cells. Another way of uh manipulating the glycosylation is to add in inhibitors of glycosylation. For example, swainsonine is very well-known compound that you can use to inhibit to inhibit the mannosidase 1. Uh and then in this way you will get high mannose N-glycans on your protein. And this is good if you want to remove them afterwards because this enzyme can can easily remove them. It's not so easy to remove the complex N-glycans, but the high mannose it's easier. And this gel here shows you the different uh migration. It's I mean, this is the same protein but produced in different cells or with or without this inhibitor swainsonine. And you can see uh here for example, in the CHO cells it's a very broadly migrating band. So, you get quite a diverse glycosylation. But [clears throat] then in the other other examples here show that uh the protein is more sharp. It's a sharp band, but they also migrate differently. So, this indicate that the glycosylation is actually different in the different samples. So, it's the same protein but different patterns of glycosylation. So, if if that is of interest for for your protein, this is something that we can also discuss and help you with to find a way of doing what you want. Okay, so that was a little bit about uh my lab Uh which is the mammalian protein expression platform. And I will now end this presentation by giving you two examples of projects that we have worked with also in the in my lab. And the first one was a project that we got from from Wolfgang. It's for the production and purification of the main glycoprotein from Ebola virus. The EBOV GP. >> [clears throat] >> So this is quite a large protein. It's the main glycoprotein of the Ebola virus. It is a large protein as I said. It also has a mucin-like domain which carries numerous O-glycans. So this is the sequence of the whole protein and in gray here you can see what the gray is the whole GP1 and the purple one here is the mucin-like domain. So it's quite a big part of the extracellular part of of of this protein. This is one gene but it's the protein is then cleaved into GP1 and GP2. And GP2 then is the bit at the end here. This protein is then also forming trimers which I will come back to. So what we did we produced this Ebola glycoprotein in 293 cells. We did this by transient transfection. With this reagent factor pro. We harvested the cells in 96 hours post transfection or I should say we harvested the culture medium because this was then produced as a secreted protein. And we did the first purification step using the His-tag on cube indigo column. And this then shows the gels from the fractions from the his purification. Uh we eluted then stepwise with increasing amounts of imidazole. Uh and we could then see the different fractions. We could see the the different or we could see where the protein ended up. So, this is a western blot specific for the his tag. And this is a coomassie gel. >> [clears throat] >> Uh and this gel was run with DTT, meaning that the because I I didn't say that, but the GP1 and GP2 is linked together by a disulfide bond. So, if you add in DTT, this will be reduced and then you see uh the two parts of the protein falling apart. And so, this is GP1 and this is GP2 down here. And in the western, you mainly see GP2 because this is where the his tag is located at the C terminus. So, you don't really see much of GP1. Could even be some uncleaved maybe up here. So, this was the first purification step. So, we uh pooled all the fractions with which contained the glycoprotein and then proceeded with the second step, which was then a size exclusion chromatography. And then it looked like this. So, it's not only one uh obvious peak here, but three or maybe even four different peaks. So, uh Yes. So, and then we ran these fractions on on gels to try to figure out what the different peaks are. >> [gasps] >> And you can see here the the how it looks in a coomassie gel. This is now non-reduced, meaning that you we only see one band because the GP1 and GP2 two will be uh still linked together. So, this is what you see in peak one. And the second peak here, and there's a small one if you remember the one here looks like this. So, they all look the same. So, they look the same on the gel, but in the size exclusion, they migrate differently. So, there must be a difference why they come out on time points from the column. The third peak, the one which is oops, sorry, the latest one actually runs like this on a gel. So, this is that was also then the question what does what that is. So, we delivered this to Wolfgang, and in his lab they made this OmniSec analysis, which is a kind of a SEC mouse where you look with different detectors to to determine the the molecular weight and also determine the the composition of the protein because this instrument can also analyze the how much of the mass that you see is protein, and how much of the mass that's actually carbohydrate. Remember that this is a gly- glycosylated protein, and it's quite extensively glycosylated. So, uh uh this and you will see that in the next slide because this is then the result of the OmniSec analysis. There is a lot of information here, but this is then the the red here is the the peak, so to speak, from the from the SEC column. Uh and the um Then you get the information about the molecular mass, the weight of the protein that comes out that in these different time points. And this is what you see here with this brownish line. Uh and then you see the composition because this the blue one here is the the part it it represents the percent of of the the weight which is actually consisting of protein. And the green one is then uh what consists of carbohydrate. You can see that this changes over time. I mean there will be a varied glycosylation. Uh so the the carbohydrate content over this peak is actually decreasing. So there is a In the end, if you pull this, there will be a mixture of different compositions. But anyway, from the molecular weight, you can say that this is then peak one and from the molecular weight, you can say that this is this corresponds to the trimer, the trimeric form of the Ebola glycoprotein. And it has a glycosylation content of well, approximately 40%. And from the mass of peak two, uh which is lower than peak one, you we concluded that this is a monomer of the glycosylated uh glycoprotein. So we had uh if you remember how it looked in the in the chromatogram, so the main peak here number two is actually the monomer and the smaller peak here is the trimer. And it turned out that the uh peak three here did not really have any carbohydrate. So that's a non-glycosylated form of the monomer. So we get different versions of of the same protein when producing it in this way. Uh okay, and this protein we delivered to Wolfgang and he is using that in a collaboration in this EU project called Vigilant, which is then a Well, maybe you can correct me now if I'm wrong, Wolfgang, but I think it has to do with protease cleavages of of these viral proteins. >> Exactly, and this to develop inhibitors against that. >> Mhm, yes. Mhm, okay. So, that was my first example. And uh, the second example uh, is a protein from TBE, tick-borne encephalitis virus. It's the non-structural protein one from this virus. Uh, this is a project that we had together with Professor Thomas Bay Strand here in Gothenburg. Uh, they are working a lot with serological diagnostics. Uh, and I mean, the main protein that is used nowadays, as I understand it, for the diagnosing TBE, is the envelope protein. Uh, but for different reasons, this doesn't always work so well. So, they were looking for on an alternative antigen for for their serology. So, we set out to produce this NS1, which is the non-structural protein uh, in in the virus. We produced it within 293 cells, and we had a His tag, so we could produce purify it using IMAC. And this is a gel then of of the protein after this one-step purification. You can see it's not super pure, but uh, for for this type of analysis, using it as an antigen in an ELISA, this is often enough. So, they proceeded to try it in their ELISA anyway. They also made an AlphaFold model of this NS1 protein. So, then presumably it looks like this. They also did some epitope detection by PepScan analysis looking where the antibodies bind, which peptides the antibodies bind to. And they could have identified two different epitopes from this analysis, which are then located here in these domains. And they continued and analyzed both serum and cerebrospinal fluid from 69 patients in this study. And they analyzed both these liquids then for IgG and IgM antibodies. And what they could find was that they got a good activity of antibodies to NS1 in both serum and cerebrospinal fluid. And their conclusion was they also checked of course specificity and so on. But their conclusion was that this is a very good antigen and they are proposing that this should be more included in routine diagnostics of TBE in the future. Okay, so I'm coming to the end. This is a picture of all of us working in PPS when we met in Lund last year. And with that, I want to thank you for listening to my presentation. I want to thank the people who have been involved in the work both in PPS and uh group in the MP core facility, and also the researchers for for their projects that they gave to PPS. So, thank you, and I'm open to questions.