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you.hello everyone and.complete workflow so.to protein it is presented by Michael.Liz PhD a senior manager in R&D and.thermo Fisher Scientific I am Judy.O'Rourke of lab roots and I'll be your.moderator for today's event we're.delighted to bring you this educational.web seminar presented by Lab roots.sponsored by thermo Fisher Scientific.for more information about our sponsor.is it WWE thermo Fisher com before we.begin I'd like to remind everyone this.event is interactive we encourage you to.participate by submitting as many.questions as you want at any time you.want during the presentation just click.on the ask a question box located on the.far left of your screen and type your.questions into the drop-down box it.appears on the screen will answer as.many questions as we have time for at.the end of the presentation if you have.trouble seeing or hearing the.presentation please use the ask a.question box to let us know that you're.having a problem please join me in.welcoming dr. Lynn I will now turn the.presentation over to him thank you for.the kind introduction and hello.everybody for this little webinar about.our complete workflow solutions from.gene to protein so my name is Michael.Liz I'm with gene arts in 16 years here.in Germany and I want to tell you a.little bit about our service which we.eventually have expanded and going.beyond a simple gene optimization and.gene synthesis to actually use these.genes to express and purify proteins in.mammalian systems for our customers.so a slight overview of the agenda I.will introduce you and give you a small.overview about what gene synthesis.actually is and what then next what gene.optimization is so the design the in.silico design of sequences prior to.their actual synthesis then I want to.give you an overview of the results of.anime lien multi gene expression study.which we did with optimized genes and.tell you a little bit about the.mechanisms of gene optimization how does.that actually work within the cell and I.give you an overview of the applications.and the benefits of optimized genes for.expression talk a little bit about a.protein expression systems at thermo.Fisher and then of course go deeply into.the gene to protein services that we.offer and then open for your questions.and hopefully my answers so what are.synthetic genes I think by now its.really a commodity it wasn't that way.when we started with gene art some 16.years ago.synthetic genes are double-stranded DNA.of course and they can have any length.they we routinely synthesize over 10 KB.genes in length and they actually can.have genome size so they are several.hundred kilobases in size the design of.synthetic genes is completely free so de.novo synthesized genes can be made upon.any specifications that the scientist.may require and of course certainly all.genes that we produce our 100% sequence.verified.now how does gene synthesis work it all.starts in silico so imagine you had take.gene from jellyfish GFP and you want to.transfer it to the fruit fly and of.course this works because the synthetic.the the genetic code is universal and.all we all speak the same language.however I would say that the dialect is.a little bit different and what I mean.with dialect this that the synonymous.codon usage in different organisms is.different and this is where we can do an.adoption in the computer and modify in.these cases here the wubble basis marked.in red to match the most frequently used.codons in the fruit fly so this is all.in silico now we have an electronic.sequence in the computer and the next.task is of course to turn this into a.tangible molecule that starts with oligo.synthesis we have apparently Europe's.largest oligo synthesis lab here in.Regensburg in germany but to date we.entirely use it for gene synthesis so.here of course we have the four anodite.say c g and t and on the machines they.are made into all egos which have.lengths something between 50 or 100.nucleotides.are designed in a way that they overlap.each other like you will see on the next.picture here the animation starts story.apparently it does not start with the.animation but anyway so they are.designed in a way that the ollie goes.they overlap each other to eventually.assemble a full length molecule that.molecule then is not covalently linked.but these missing gaps of the.oligonucleotides are filled in with.polymer races and after a few cycles of.cycling basically this primer extension.reaction leads to full-length molecules.which can then be amplified sorry now.you will see the animation so the ollie.goes they find each other the gaps are.filled in with polymer races and you end.up with a full length molecule which can.then be amplified and cloned into a.suitable vector transformed into e.coli.and colonies can be picked to verify the.correct sequence of a monoclonal.molecule that's basically how gene.synthesis works and the overall process.depending on the workflow that you use.may take about five days.synthesis has become true commodity over.the years and to date we have many many.customers that don't really bother.cloning anymore and amplifying a.sequence from some template in the.fridge using primers to modify.restriction sites and so on but they.just order this sequence that they have.in the computer anyway they are.synthetic genes are today used in all.kinds all areas of biotechnology like.basic research they used in functional.assays for antibody production for.protein production actually also for.optimization of proteins and enzymes if.you adapt synthetic genes to directed.evolution which is possible you can use.synthetic genes as positive controls for.DNA detection essays you can use them.for cell and optimization actually for.anything you can think of where you need.DNA.so in this picture this is kind of the.portfolio that we currently offer within.synthetic biology at thermo Fisher gene.art of course we help our customers to.design projects according to their.specific needs we do gene optimization.and gene synthesis which I just.explained a little bit and we can make.gene variants if you for example want to.do an alanine scan and require several.hundred or of different similar.sequences we can do this as well we can.make gene libraries for page display for.example creating huge diversity in these.synthetic genes sub cloning is actually.a big part of our portfolio because we.have many customers who say yes I want.the synthetic gene but please provide it.in this and that vector then plasmid.production up to milligram levels and.then also cell lines and proteins which.I'm going to focus on now.I want to explain a little bit what gene.optimization is and how it works so as I.mentioned before biotechnology is.possible or is actually easy because the.the language of all the organisms is.basically the same and it's easy to.transfer a gene from one organism to.another and have a good chance that it.actually works however here's a small.part of some codon usage tables of four.organisms a plant human yeast and e.coli.and if you look at the six synonymous.arginine codons and look at the columns.of humans and he coli you will notice.that humans with the blue shaded numbers.prefer some different codons than e coli.the blue shaded codons and vice-versa.the rare codons in E coli are the most.frequently used codons and Homo sapiens.and so on that means if you for example.take an insulin gene from a human and.plant it into bacteria it will work.however it will work with not the most.frequent codons and that may have an.impact on the expression levels of that.specific insulin protein.you.that's about codon usage codon usage is.not the only parameter in the dialect of.the different organisms I would say.there are other parameters that can be.adapted as well and that's for example.GC counter content.everybody knows that different organisms.have a different GC content and it's a.good idea to adapt the GC content to the.host in order to facilitate or to.guarantee genetic stability good.performance and also good expression.then for example cryptic splice sites.may be an issue if you go from if you.express in a human in mammalian cell it.may be a cDNA or gene from another.species then it may happen that there's.a cryptic splice site and the mRNA is.not processed correctly and you end up.with zero expression there's actually a.very famous example when they first try.to express jellyfish GFP in tobacco they.got zero expression and they found that.the jellyfish cDNA contains a cryptic.splice site which is recognized by the.plant and the DNA the RNA is not.processed correctly premature.polyadenylation signals may also be an.issue in 40m mrna design or iya design.direct repeats in the in the DNA may.lead to instability netic instability in.direct repeats may lead to RNA secondary.structure which also hamper translation.and then for example in plants their.so-called killer sequences that's a tea.rich sequence or sequence stretches that.have a impact on the mRNA half-life and.lead to a faster degradation of the mRNA.so all these parameters can be taken.into account when you optimize.gene in silico now optimization of gene.and silikal is actually not that trivial.and that is because the number of.sequences coding for one and the same.protein is astronomically high it's the.number of combinations of all the.synonymous codons so how did we solve.that that problem and I show you here.short animation of how our algorithm.basically works so you see that our.album algorithm for this short peptide.would come up with the most preferred.codon for almost all amino acids but.that 3:09 is coded by the second most.frequent codon for what reason in this.specific case I don't know maybe the.algorithm said okay I rather use ACA.instead of ACC because a cc would push.the GC content over a certain threshold.or splice site would be generated or.maybe a restriction site would be.generated which should be included and.so on so I mentioned that the number of.combinations of different codons is.astronomically and how does our.algorithm solve that it's a sliding.window approach so imagine that in the.first row you see a small sliding window.covering six different codons and for.these six codon positions it generates.all different or all possible.combinations and chooses the best one.fitting to all the parameters GC content.most frequent use codon no splice sites.and so on then the first codon is fixed.the sliding window moves one one amino.acid further and it does the same thing.again.then the first first codon is fixed and.so on so and that way it slides over the.complete window the complete open.reading frame that's our approach and it.leads to a sequence which uses the most.frequent codon as much as possible but.it also takes all the other parameters.into account.now about protein production next thing.many scientists when they think about.protein production think about a coli.because e.coli is fast it's easy.handling it's cheap well studied but.expression in e.coli has a number of.disadvantages it has no pros.translational modifications no membrane.environment for membrane proteins it.often leads to miss falling and.aggregation and inclusion bodies it has.some restrictions to large size or early.chimeric proteins and of course no.process inflation so also no.glycoproteins are possible Amalia of.course circumvent all these.disadvantages of e.coli that has it's.very own obstacles sometimes when alien.expression has some transcriptional.silencing the mRNA may be unstable in.the cell alternative splicing may happen.premature pollination or congenital.translation is inefficient and all these.things they can be overcome in to a good.design of the gene before you synthesize.it we have actually tested this in a.study a couple of years ago which we.have published and you will find the.reference and a few slides we have taken.50 genes 50 human genes and have.synthesized them as a wild-type version.and have optimized them and synthesized.them in an optimized version you may ask.ok why optimize human genes for human.expression because we then took these.into human cells and express them but of.course our genes in our body are not.optimized for highest yield but they are.optimized for highest performance in the.overall context of the cell so.expression maximum expressions of course.never a goal of almost never goal of the.evolution and the design of our genes so.here's some snapshots of the outcome all.the transactions and quantifications on.the Western blot were done in triplicate.and you see for a crept one a.transcription factor and the top row.that it's in notice blue on the Western.blog very nicely that the optimized.version had a higher expression than the.wild-type and in that case it's a 2.5 a.2.8 fold increase of expression and then.the same is true for the other ribosomal.protein for protein kinases for membrane.proteins for cytokines so on the top row.il-2 we actually did not detect any.expression when we transacted the.wild-type a cDNA but with the optimized.we got a faint band so in that case it's.from zero to something expression and.overall for the 50 genes we observed.that 996 percent of the cases for these.50 proteins we got an equal or a higher.protein expression actually in 86.percent the optimized version gave a.significantly higher expression than the.wild-type and ten percent it was equal.and only in two cases or four percent.the optimized gene expressed less than.the wild-type.so what that means is overall you get a.higher expression but also the.reliability that you get a detectable.expression at all is much much higher.overall we got of 24 25 fold increase up.to 25 fold increase in protein yields.and only two of those cases were.actually.did perform less than the wild-type.version.you.so all these tests were made in two nine.three cells and we wanted to see whether.it we get the similar results in Chinese.hamster cells and in SF nine insect.cells and basically you see that with.the green arrows the results were very.comparable so op gene optimization.worked equally well in 293 and in cho.cells and in one case in the insect.cells it was no in there hector and.three cells it was less and india even.in the SF nine cells the expression rate.was higher as compared to the wild type.versions.you.now how does what what's the mechanism.behind gene optimization I explained the.parameters codon frequency GC content.but what's the conceptual what's the.concept behind optimization and why does.it lead to a high expression we have.tested in this case for mid 1 alpha.several parameters in the transfect.itself so you see obviously it has an.effect on the overall expression level.of the protein in that case with the.optimized version we got a three-fold.better expression as compared to the.wild type if we looked at the de novo.synthesis rate so the nuclear run-on.rate of transcription we also find found.a 30% increase of that particular step.in in the overall expression or.translate transcription and translation.context also if we looked at the mRNA.stability we found a 14% increase of of.the stability of mRNA probably due to a.slightly higher GC content as compared.to the wild type.um.then on a northern blot we found that.the optimized mRNA did not perform any.better when it comes to nuclear export.so wild-type and optimized mRNA were.equally well exported from the nucleus.into the cytoplasm and the mRNA steady.state levels were 18% higher.that's probably an effect that comes.from both from the de novo synthesis.rate so the nuclear run-on of the mRNA.and the mRNA stability in combination.gave 80% higher mrna abundance inside.the cell and overall of course the.translational efficiency was 20% higher.so that means that the mRNA once it is.there the efficiency of turning this.into a protein on the ribosome was also.20% higher as compared to wild-type.maybe due to the lack of any or the.elimination of secondary structures in.the mRNA.hmm when we optimize genes for our.customers we often get the question yeah.okay I see that the expression rate can.be increased but will the final protein.be functional I tend to answer that this.is a general question in biotechnology.whenever you have a gene from one.organism to transfer it from one.organism into another then you are in a.I would say codon context that is not.natural in terms of it has adapted by.evolution so but in this case and so.that's it's the same thing whether it's.from another organism and coming into a.cell or whether it's synthetic coming.into a cell and it's not there's no.conceptual difference however of course.you can argue if protein is expressed.really high then maybe it lacks the time.for proper folding and may lead to a.larger proportion of non-functional.protein so I will show you here an.example of three proteins kindnesses in.that case which we expressed in also.again coming from wild-type DNA or from.optimized cDNA and for junk I and they.all expressed better were from the.optimized genes and for Jan kinase one.we did prosper relation si and in a.nutshell what we found is that the.protein coming from the wild-type gene.of course was able to give a positive.kinase signal but this Jean kinase.coming from an optimized gene ahead the.very same.which you see on the very right side.with a phosphate specific antibody so.there was no difference in that case we.could show that both proteins or the.same protein coming from both genes had.very comparable function another.interesting thing that you can do with.optimized genes now the optimized gene.has a different sequence than the.wild-type gene that means that the.optimized genes will react differently.to an SI RNA than the wild-type gene so.if you design si RNA against the.wild-type gene it's unlikely that or you.can design the optimized gene in a way.that D si RNA will not silence the.optimized gene and we did this with.cyclin dependent kinase 2 which is a.cell cycle regulator and you see on the.so and the facts plot on the top left.this is the untracked itself as usual.most cells are in the g0 g1 phase and.only few cells are in the g2 2 phase if.we transfect a nice i RNA against the.wild-type cdc2.then more cells are arrested in t2 now.if we transfect this SI RNA together.with an optimized gene for cdc2 then.cdc2.can be expressed again and this si R&A.cannot silence the optimized version and.on the bottom left you see that again.the fraction of cells in arrested in g2.decrease.so that's a very interesting function to.or feature of optimized jeans to to.validate si RNA functionality so the.summary of the study is that we could.show with 50 genes that gene synthesis.and gene optimization really increases.the overall expression and the success.rate of successful protein expression it.improves general yields and it does not.alter the solubility or the activity of.encoded protein and allows to confirm si.RNA phenotypes.now there are of course other companies.out there competitors who also offer.gene synthesis and gene optimization and.we have taken a closer look about.different optimization strategies so in.silico optimization strategies some of.them are very different from ours and.some of them are similar to ours I would.say we have synthesized such genes from.different competitors designed from.different competitors and tested them in.a mammalian expression analysis here and.so in a nutshell what you see here is.that again the gene art optimized genes.at a leaf about 3 fold higher expression.than the wild-type gene but then there's.also expression levels from genes.optimized from competitors and they are.all or expressed less than than the gene.are not just the gene art-design gene.but even less than the wild-type gene.which of course is not very favorable.because you typically want to increase.and not decrease your expression.so on this slide what I just want to.focus you on is that the protein service.that we offer is not the only service.that we have in our portfolio around.gene synthesis there's many other.products which most of you are likely to.be familiar but here and this particular.talk should be focused on the proteins.and expression in heck intro ended.insect self.poor Dean's are for the expression we of.course rely on the excellent reagents.that thermo Fisher allows us to use and.that is of course free style and since a.year for Joe or since three years four.to nine three cells the XP system and.what does the service look like there's.different kinds of requests from.customers and we have shaped our.services around these different needs.from customers so if you would need a.protein to be synthesized we need from.you and amino acid sequence.electronically or an accession number.and that's basically it but then you.also need to specify on your.expectations so the yield that you need.this can be specified culture volume you.may say okay please deliver whatever you.get from one liter culture and you can.of course also specify which cells you.prefer whether it's two nine three or.cho cells or insect cells and in that.case you would of course get the.synthesized gene in an expression.plasmid you would get the purified.protein from that specified culture.volume or you may also get the rare raw.culture supernatant or the cell pellet.in if you need a specified protein.amount then that's also possible also.with the same cells and with the same.deliverables but we need to do a gene to.protein pilot first so what we would do.then is we make a small culture volume.transfect that expression construct do.the expression also do a time course.about the harvest day do purification.and then we know what yield we got from.that particular culture volume and can.then upscale accordingly to meet the.specified protein amount that you.requested.you.so next I want to talk a little bit.about some customer success stories some.show you some examples on how gene.optimization and gene synthesis and in.conjunction with the XP systems works or.gives a better performance for our.customers this first example is still.without the XP system in that case we.made all nine combinations of three.different light chains with three.different heavy chains for a customer.and the customer already gave us some on.the top left table you see a gave us.some expression yields that he was able.to achieve and on the right hand table.you see what expression levels we.achieved after we optimized the genes.and expressed that and down there you.see the same results also in a bar so.the overall increase was about 20 to 50%.50 fold and in one case even 650 fold.increase.you.II.Joe was different iterations I would say.of combinations of gene vector and.production cell line so different aunt.if six different antibodies from a.customer and the white bars and the.graph represent the wild-type gene and.customer in-house production those.expression levels and then.dark blue bars are already optimized.gene but in a customer vector and with.the freestyle ternary self so already an.increase and the gray bars is optimized.gene man now in pcdna 3.3 vector and in.a freestyle system in some cases for.antibody 3 and 4 that gave a nice.increase and then the the right blue.bars are then everything optimized.optimized gene PCD and a 3.3 and XP 2 9.3 which in almost all cases gave much.higher yield of protein expression for.the sake of time I will touch this only.very loosely because I have some other.so here's some antibody expression.results comparison of XP 2 9 3 versus XP.Cho and basically shows that both.expression system work remarkably good.and give well folded antibody you see.that in the chromatogram HPLC plot down.there and heavy and light chain are well.folded and into a full functional.antibody.this is what doctor.of a protein looks like it's a little.bit hard to read I think on a small.screen however what I want to say here.is that you get a for our customers get.full documentation about all the aspects.that were used from gene synthesis to.protein production and purification so.starting from the sequence of course is.documented and then which vector is used.with which expression cells the culture.volume how it has been purified if ended.what the endotoxin levels are of the.protein the concentration how it looks.on a Kumasi gel what the HPLC.purification looked like which fractions.have been pooled for the protein and.with a final chromatogram of the pure.then purified protein.this documentation is highly.customizable and is usually discussed in.advance with a customer and each and.every project.here I can show you how easy it is to.order proteins mm synthetic proteins so.on the thermal website actually it's.easiest to just google gene synthesis.thermo Fisher you find a gene art gene.synthesis tab and if you click on that.there's a link to gene arts cell lines.and proteins and on the website that you.reach then there's a small download.button that allows you to download a.request form which you can then email to.the given address that's basically it.and then that form you can enter of.course the sequence or the accession.number and what purification method you.need what yield you need and so on so.already going through the overview what.I hope I was able to show you is that.with our gene optimization we can.increase significantly the protein yield.in the mammalian system of course also.in other systems but here we're focusing.on mammalian expression there's full.design flexibility if you want to use.gene further and require specific.restriction sites or Gibson cloning site.yeah.designed for Gibson cloning then this.can all be included in the design we of.course used in vitro gen vectors huge.that's compatible with the XP system we.have high quality plasmid preparation in.order to basically to minimize endotoxin.evel's in the protein and the whole.process is done in-house and that of.course allows for best turnaround time.from the electronic sequence to the.purified protein.and yeah I don't want to read that out.loud we express in to line three cells.in XP and Freesat style system and also.in cho cells and in back-to-back sf9 SF.21 and high five systems we of course.purify the proteins on F plc an HPLC.on different columns which are of course.project specific and upon customer needs.we analyze the proteins that we.synthesized with Center page 280 nano.meter a quantification also with a blitz.if it is a an antibody with HPLC and we.are just setting up a mass spec then so.that will be part of on an analysis QC.as well.so the gene to protein I said in record.time the overall gene to protein service.typically takes 30 business days but can.be faster in times that really depends.on specific project like the size of the.gene and the required yields of the.protein and so on the performance is.extremely reliable our gene synthesis.reliability is about 100 percent and.then it comes to proteins there are the.results a little bit more varying but we.usually almost always get measurable.expression but of course that's very.much protein dependent expression.protein expression is extremely scalable.and very flexible so we produce from 100.milliliter cell culture volumes up to 25.liter wave back volumes.and the whole system is very secure.of course the whole e-commerce system is.iso certified and of course our staff is.trained also accordingly and with that I.hope that I have given you some overview.of our gene to protein services at.thermo Fisher gene art and I'm happy to.take some of your questions if you may.absolutely thank you dr. list thank you.dr. list of an informative presentation.we will now start the live Q&A portion.of the webinar if you have a question.you'd like to ask please do so now just.click the ask a question drop-down box.located on the far left of your.presentation window type your question.into the box that appears on your screen.and click the send button well answer as.many of your questions as we have time.for let's get started our first question.is do you recommend incorporating signal.sequences in the construct to express.membrane proteins if it's a membrane.protein then of course it already has a.signal sequence if it's a mammalian.membrane protein that is we do an.analysis of the sequences prior to the.design if we detect a single sequence.then we know it's secreted or ends up in.the membrane if it does not have one.then it's in this we discuss with a.customer whether you want it to have it.secrete it then we can add an artificial.signal sequence in order to secrete it.or if it comes from another organism and.should end up in the membrane to end up.in the membrane okay thank you what is.the major factor at the level of.transcription for a non expresser or.poorly expressed protein.can you repeat that question please yes.what is the major factor at the level of.transcription for a non Express ER or.poorly expressed protein hmm I mean.that's a hypothesis that it's if it's.poorly expressed that it is due to low.transcription I would say my best answer.is of course it's a matter of a good.promoter in order to have a high a good.transcription second is that the 5 prime.5 prime end of the mRNA should be.somehow relaxed maybe a little bit baby.a little bit a team or a tea rich in.order to facilitate transcription but.then I think it's it has but what has.more impact on the level of expression.in terms of mr or RNA is not so much the.transcriptional activity but more the.stability of PM RNA in the nucleus so if.you think about cytokines for example of.course they are expressed extremely well.upon inflammatory signal for example in.our cells but they should be expressed.only for a short time and therefore they.have a very short half-life the mRNA.have a very short half-life so that this.protein concentration diminishes quickly.enough and the cytokines make this.possible due to specific a T rich.regions in the mRNA.thank you apart from codon optimization.does Kozak sequence have any major.effect we typically include a cosmic.sequence I personally and and that's.that's a little better religious.question I personally don't believe more.I haven't seen a much much convincing.data that the Kazakh sequence really.helps we include it because still it.appears quite a bit in the literature.that its state-of-the-art to include a.cosmic sequence and for us it's easy.yeah it's a few nucleotides more and.doesn't add to the cost and it doesn't.harm at least so we typically add a.Kazakh sequence but we haven't seen.convincing evidence that it actually.helps the next attendee says thank you.and asks is the optimization free or.connected with an order.again can you repeat that please.yes is the optimization free or.connected with an order free.great simple as that how long did it.take to receive a protein and what is.your turnaround time yeah that depends a.little bit on the overall scope of the.order so in the for the most.comprehensive service you send us a.[Music].sequence electronically we synthesize.the gene do a larger scale a plasmid.preparation transfect a liter of cells.and incubated cells for six seven days.then harvest purify polish the protein.to the fill and finish and the.quantification that takes about 30.business days it can be faster if the.scale is lower or sometimes customers.say I have the expression plasmid.already I'm going to send you that so we.can skip the gene design and the.synthesis gene synthesis part but can.start right away with plasmid.preparation and with transfection then.it's shorter but I think around about 30.business day is a very good number to.start with this next one is a two-parter.I'll read both parts in case it's.helpful and then let me know if you need.me to repeat the second part do you also.code on optimize the reader sequence and.is it sometimes better if the.translation in the leader holds for.allowing a better folding yes we do.optimize the leader sequence and we have.no observations that it up to.optimization hampers in any way folding.or functionality of the protein I would.say that 80% of the proteins that we're.making are actually antibodies and of.course here the demand is that the.expression rates are as high as possible.in the yields.and so we use the yeah we have never.seen that antibody lacks folding or.proper phenotype upon optimization of.the leader peptide okay thank you the.next one is when should somebody prefer.insect cell expression over mammalian.cell expression that's a good question I.think I can't give you good answer to.that because I personally haven't worked.with insect cells before from what I.know though is that some proteins.Express extremely well in insect cells.and of course the nice thing is that if.you choose the insect expression system.then the first thing that you do is you.generate virus baculovirus and with that.you infect the cells so that is kind of.a Perpetua stiff source of of.reinfection of new cells so it's once.you have the virus it's very easy and.very fast to make new protein batches.this I see is one of the advantages and.then from what I've heard is that some.proteins just Express extremely well and.insect cells do you incorporate five.prime UTR for protein production.purposes apart from the Kozak sequence.usually not there are within the five.prime UTR are within the vector and the.PC three-point-three that preferred for.the XP system already contains all these.parts which is essentially a really.strong promoter and other than that.nothing else is necessary.which purification which purification.methods do you offer including epitope.tags hmm so routinely as I said we 80%.of the proteins that we're doing are.antibodies of course these we typically.purify with the FC with their sea part.with protein a and gel filtration for.polishing but we also use other tags.like histep tag strep tag flag tag or.other customer specified tags we can.also leave the tag up on tags upon.expression here we usually use teff but.can also use other proteases for.cleavage of the tags.do you offer membrane protein.purification no membrane provocation is.a very specific I would say art already.and it can become very tedious and very.protein specific we do offer membrane.protein expression but in these cases we.typically deliver only the cell pellet.but of course we verify the expression.with the Western blot but then leave it.up to the customer to use the cell.pellet for further use of the membrane.protein.what is part of your QC or what is.included in the documentation hmm.I shown it documentation before so yes.for the protein it's a standard Kumasi.gel of denatured and native protein then.the concentration as measured by OD 280.Western blot then for antibodies we.usually do a protein a based blitz titer.determination and then optional are.other QC methods like HPLC or an endo.tox test is also optional or mass spec.things like that how much protein will I.get if I order your service it's highly.dependent on the protein itself of.course so if you order the volume option.which is please transfect a liter of.culture and get me whatever you can.express from that then it's open what.the expression levels will be so from.antibodies I can tell you that typically.from a liter of transfected cells we.achieve 100 to 300 milligram of antibody.but there's already also depends on the.antibody itself but if you say no I want.100 milligram of this and that protein.then we have to do a pilot first and.then second scale up on volume according.to the results of that pilot we have.time for one more question.I am surprised to see your results that.code on optimization from different.vendors work very differently major.differences.that's not really a question yeah but I.I hear I can I can understand that.surprise and I would this is this is.kind of a showcase but we have done.these studies for other proteins as well.and we see maybe not that extreme but we.see very similar results so you can make.that experiment on your own you can go.to different vendors and do the.optimization on their website and see.the results you can hit the optimize.button several times and from some.vendors with their algorithm you will.see that every time the sequence will be.different when you optimize it so it's a.stochastic algorithm that's one thing.that would make me suspicious what you.can also do you can take a sample.sequence optimize it at different.vendors and then look at the GC content.and you will see differences in the GC.content and then if you look at what is.the optimal GC content for mammalian.expression just by looking at the.sequence or the GC content you will.observe that some of them may have a.higher likelihood to express better than.others so this is an experiment I would.suggest you to do if you're interested.to dig into this to get more confidence.and in this but I really can assure you.that the results that I have shown you.may be extreme art but are kind of.representative from what we observed.with many different other proteins as.well well I would like to once again.thank dr. list for his presentation I.would also like to thank Cyril Thermo.Fisher Scientific for making today's.educational webcast possible before we.go I want to let everyone know that.today's webcast will be available for.on-demand viewing through May of 2019.please share with your colleagues who.may have missed today's live event also.stay tuned for our next presentation.cryo-electron microscopy.revolutionising the world of structural.biology and healthcare it is presented.by mark MH storms a product marketing.manager in the life sciences business.unit of the materials and structural.analysis and analytical instruments.group at Thermo Fisher Scientific you.won't want to miss it that's all for now.thank you for joining us we hope to see.you again soon goodbye.thank you goodbye everybody.

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