Bacterial expression and ligand binding of purified domains of Stabilin-2

Lauren Vatter Author

08/01/2021 Added

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This summer I worked to characterize the ligand binding on the Stabilin-2 receptor found in the liver.

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[00:00:00.270]And it was Lauren and I will be taking you through, um,
[00:00:03.810]my you care project this summer, which is entitled that bacterial expression,
[00:00:07.380]like in binding a purified domains to build.
[00:00:09.530]And to I focused on this a lot this summer,
[00:00:13.050]I did a little bit of research on this towards the end of last semester,
[00:00:17.430]but this is the main focus of my research this semester or
[00:00:22.410]the summer I should say.
[00:00:24.360]So basically I'm going to start with a little background information
[00:00:27.360]introduction about why we do this research. What's the significance of it?
[00:00:31.590]So heparin is a very important anticoagulant used in hospitals and outpatient
[00:00:36.060]care. It's one of the top 10 drugs that's used in hospitals.
[00:00:39.870]So it's very prevalent in medicine today. We,
[00:00:43.200]it's very important that we know how it interacts with our body.
[00:00:47.160]So there are two different forms of heparin,
[00:00:48.990]the unfractionated and the low molecular weight heparin unfractionated heparin
[00:00:53.100]is used more in hospitals because medical professionals have to really control
[00:00:58.080]the dosage of that.
[00:00:59.250]And then low molecular weight heparin is basically what it sounds like.
[00:01:03.120]It's a smaller, um, molecular weight. It's basically fractionated heparin. Um,
[00:01:07.620]so it's the, uh,
[00:01:09.450]heparin molecule is just split apart and that is more used in
[00:01:14.040]outpatient care because it does not need, um, constant supervision
[00:01:19.500]of dosages.
[00:01:21.000]And so antisense Akio nucleotides also known as Osos are
[00:01:25.830]also a drug that's used in drug therapies to correcting splicing
[00:01:30.780]and for knockout genes and, and receptors.
[00:01:34.230]Catabolize both ASSP and heparin.
[00:01:37.200]There are two stability receptors to build on one and stubble in twos to build
[00:01:40.500]in to, um,
[00:01:41.640]does most of this catabolism patient interacts with these drugs such as ASO and
[00:01:46.560]heparin. So that's what we used in our research.
[00:01:50.100]That is what we study stability to.
[00:01:52.200]And stability two is a 315 kilodalton receptor.
[00:01:56.940]Um, but it can also be cleaved into a 198.
[00:02:01.200]Kilodalton variant known as one 90 hair and hyaluronic acid is the
[00:02:06.090]first known ligand that binds with [inaudible] and hyaluronic acid is important
[00:02:10.590]when we assess the ligand binding and our research that I will talk about a
[00:02:14.550]little bit later.
[00:02:15.990]And this graphic here is just kind of showing the stubble and receptors.
[00:02:20.550]So up here, we have stubborn one,
[00:02:22.800]the receptor that we don't really study that doesn't, um,
[00:02:26.580]it interacts with heparin and ISO's, but it does it to a lesser extent.
[00:02:31.230]So here we have stubble into,
[00:02:33.810]this is the 315 kilodalton variant, obviously it's the longer one.
[00:02:38.970]And then under we have one 90 hair.
[00:02:41.820]So the interesting thing about stability, um,
[00:02:44.880]the 315 killed on very enough stability in one 90 hair is
[00:02:49.500]that, um,
[00:02:50.400]it binds at 19 ligans and all of the ligans that bind to a
[00:02:54.990]stubble and to also bind to one 90 hair.
[00:02:57.990]So it's been concluded that all of the binding
[00:03:02.830]happens in the latter two thirds of the receptor.
[00:03:06.050]So basically this part does not bind to any ligands.
[00:03:09.040]It's just this part right here.
[00:03:14.770]So the objective and purpose of this research, so stability to, as I said,
[00:03:19.570]binds 19 different logins,
[00:03:20.980]but only two of those ligands have been characterized.
[00:03:23.320]So what about the other 17? That is what we're researching.
[00:03:26.740]So we're researching what region of spilling two interacts with these
[00:03:30.130]electronegative polymers and the characterization of those logins,
[00:03:35.470]and do that through a six step methodology, um,
[00:03:38.890]to evaluate this ligand binding. So we go through PCR transformation,
[00:03:43.510]the verification that we transform the correct plasma and the protein
[00:03:47.110]expression, protein purification and ligand binding, which can be, um,
[00:03:51.700]outlined here in this little graphic.
[00:03:54.370]So I'll explain each of these methodologies in more detail.
[00:03:58.720]So first we have PCR. So we start with PCR.
[00:04:03.850]So we anneal C3 and C4,
[00:04:07.060]which is basically just shorthand for the third and fourth EGF
[00:04:11.500]clusters on the one 90 hair receptor.
[00:04:15.940]So we nail those and we like get them into a pet 43.1
[00:04:20.470]factor. And we also work with an empty pet vector. And I also, this,
[00:04:25.710]this summer, I heavily worked with the link domain as well.
[00:04:31.000]So once these, uh, [inaudible] these,
[00:04:35.410]the link domain is ly gated into the pet factor.
[00:04:39.940]All of this recombinant protein has a six S his tag,
[00:04:42.550]which is important for the protein purification that we'll talk about later on.
[00:04:47.560]So the next step, well, first I'll talk about this, um, gel right here.
[00:04:51.790]So basically we have in this gel,
[00:04:55.180]I annealed to different parts of the
[00:04:59.710][inaudible] EGF Z four, excuse me, as you for EGF cluster.
[00:05:03.550]So we have 6, 6, 7, 8, 6, 7 5 4 here,
[00:05:07.360]which is basically just different places on this cluster.
[00:05:11.080]And then we have the link domain here, as well, as I said,
[00:05:13.240]I worked a lot with that this semester. And so, um,
[00:05:17.200]I ran on the gel to see where they were after I ran a PCR on them and then cut
[00:05:21.910]them out. And then we moved onto the transformation. So,
[00:05:26.650]and transformation process. It's basically exactly as it sounds,
[00:05:30.310]we transformed the plasmids into Nikolai and we run them on gel to verify the
[00:05:34.540]presence of inserts here. We have, um,
[00:05:39.220]uh, big gel that I ran,
[00:05:41.060]I believe it has 18 different, um,
[00:05:46.120]colonies that I'm looking at 18 different plasmids that I'm looking at. Um,
[00:05:50.260]and sadly, this gel was, uh, pretty unsuccessful.
[00:05:55.210]The inserts did not really work. They weren't, um, my gated properly,
[00:06:00.830]they just didn't work out, but the last two here are, um,
[00:06:05.030]empty pet doctors. And so, um,
[00:06:08.180]those actually did run as they should.
[00:06:11.270]So I was able to just cut out the empty pet vector
[00:06:15.920]and a mini prep that, and work with that for a little bit.
[00:06:20.930]Um,
[00:06:21.320]so then the next step in the methodology is to verify that we
[00:06:26.240]have the correct classmen. So once, um, this, the previous side,
[00:06:30.770]this gel was just one of the many gels that I ran, um, to, um,
[00:06:35.510]verify the plasma it's free there.
[00:06:37.370]And so once I actually do get inserts that work,
[00:06:40.280]we have to verify that it's the correct plasma.
[00:06:42.980]So we do that through the restriction enzyme digest.
[00:06:46.520]So I mainly worked with bam, H I and eco I this summer,
[00:06:51.530]just cause we know exactly where that cuts in the, um,
[00:06:57.200]receptor. So I was able to cut them. And this is just an example of a job that,
[00:07:02.180]um, I ran that has,
[00:07:04.130]I believe it's still with 6, 6, 7, 8, 6 7 5 4, and then link it again.
[00:07:09.140]So we just wanted to ensure that they were actually the right size of plasma and
[00:07:13.880]everything was correct there.
[00:07:15.080]And once that is verified through the restriction enzymes digest that all the
[00:07:19.160]fragments are correct. We send the DNI off to be sequenced,
[00:07:23.120]to ensure there's no view mutations like a frameshift or drop out or anything
[00:07:27.230]like that. So once all of that is verified that, um,
[00:07:31.250]everything with the plasma and the DNA is correct,
[00:07:34.550]we move on to protein expression. So protein expression is,
[00:07:39.230]um,
[00:07:40.100]where we transform the plasmids into BL 21 or
[00:07:44.930]gummy Ecolab. So
[00:07:49.270]beautiful 21 cells are a special type of Nikolai because they
[00:07:53.980]are specialized for protein, folding and expression.
[00:07:57.550]So it just gives us a better shot of the protein being expressed correctly and
[00:08:02.440]fully when we transform them into these origami cells.
[00:08:07.090]So first this is kind of a three-day process over here.
[00:08:10.780]We have a gel of when we transform them into these origami cells and the gel
[00:08:15.700]itself didn't really turn out great. It's a little bit blurry. Um,
[00:08:20.080]I ran it multiple times and it kind of was still this blurry, um, thing,
[00:08:24.160]but we were able to verify that the, uh,
[00:08:28.390]plasmid was there. So we were able to first grow them up in a small culture,
[00:08:33.430]about three milliliters. And then once that it's grown up,
[00:08:37.870]we grow it up into a one or two liter culture overnight.
[00:08:42.700]And then finally we have an induced culture when we induce it with IP TG.
[00:08:48.100]So when we're working with [inaudible] the three pet,
[00:08:52.540]we induce them all with IP TG,
[00:08:55.260]and then we have to purify those proteins from the big
[00:08:59.520]cultures. So that there's two ways to do that.
[00:09:04.020]And I primarily only did French press. I only did French press.
[00:09:08.850]So, um, that's the only one. All we talking about, we can also use on occasion,
[00:09:12.930]but I did not do that this summer.
[00:09:15.060]So basically the French press is a machine where that applies a lot of pressure
[00:09:19.050]and breaks the cell open. So all of the genetic material comes out the DNA,
[00:09:23.760]the protein that we want. Um,
[00:09:26.790]so the proteins then run through a nickel resin,
[00:09:30.960]and this is where the success his tag comes in because that basically gets stuck
[00:09:35.160]in the nickel resident through interaction.
[00:09:37.320]And the protein is stuck in the resin and it's washed with a
[00:09:41.940]solvent to purify it. And it can either be, um,
[00:09:45.360]it can either remain in the column or be alluded.
[00:09:48.180]And this summer I alluded the proteins,
[00:09:51.930]I alluded them all. So, um, the next step is look in binding.
[00:09:56.370]That's what we're testing for.
[00:09:57.810]We want to see how well does the protein that we
[00:10:02.040]purified bind ligans.
[00:10:04.920]So there are two options to determine ligand binding,
[00:10:07.620]but I only did one this semester. I only did colorimetric assay's specifically,
[00:10:11.850]I did one BCA assay. So, uh,
[00:10:15.360]basically the protein is alluded from the column just using on the elution
[00:10:18.690]buffer. And then, and anybody is used to mobilize the protein.
[00:10:22.770]I believe we used a rabbit antibody this summer,
[00:10:26.040]and then the presence of the login is used, uh,
[00:10:28.830]is measured using a colorimetric assay. Like I said,
[00:10:31.500]I used a BCA assay and, um,
[00:10:34.380]basically the amount of color that, um,
[00:10:39.300]is shown in each sample can tell us how much ligand
[00:10:44.190]is bound to that.
[00:10:46.350]So I only did one BCA assay just because it takes quite a
[00:10:51.300]little bit of time to get through all these steps to make sure everything's
[00:10:53.940]right, and then you have to go back and we do it if it's not.
[00:10:56.580]So I did one BCA assay this summer to assess the ligand binding.
[00:11:01.170]And these are the results from that, which makes sense.
[00:11:04.620]There should be a little bit more ligand binding than this,
[00:11:06.900]but the ratios make sense. And that is the four is the most, um,
[00:11:11.580]has the most with, uh, 10 micrograms per milliliter.
[00:11:15.480]C3 is next where it's kind of stuck in the middle with, um,
[00:11:19.710]seven micrograms per milliliter and then pet, which is just the empty vector,
[00:11:24.510]like I said, has six micrograms per milliliter. So the least amount.
[00:11:28.740]And so in conclusion, and the further directions of this experiment,
[00:11:33.780]a benchmark for my research was being able to
[00:11:38.520]complete the methodologies independently and proficiently.
[00:11:42.480]And I am happy to say that I was able to complete almost all the methodologies
[00:11:46.740]independently. I was able to,
[00:11:48.660]I did all of the methodologies I ran through with this experiment many times.
[00:11:52.680]Um, I did one VCA asset, like I said,
[00:11:56.860]but I had help from Dr. Harris with that.
[00:11:59.320]So I can't say I did that independently,
[00:12:01.420]but I believe that it's a great benchmark, um,
[00:12:03.850]a great show of my progress and growth through you care that I was able to do
[00:12:08.710]these, these processes independently, especially French press,
[00:12:12.700]because I know it's not a common thing for undergraduates to be able to do that.
[00:12:16.960]And I was able to do that multiple times independently,
[00:12:20.200]which I think is very cool. And so, uh, further directions.
[00:12:24.790]So purified proteins that went through the nickel resin and
[00:12:29.350]everything were incubated with radioactive hyaluronic acid to quantify and
[00:12:32.740]Lincoln ligand binding, which like I said, um, hyaluronic acid was the first,
[00:12:38.020]um, ligan to be discovered that bond bound to the stimulant two receptor.
[00:12:42.540]And obviously I am not qualified to be really handling radioactive
[00:12:46.980]material.
[00:12:47.560]Seeing I'm as I am junior in college.
[00:12:50.040]But I just, my Dr. Harris stepped in.
[00:12:52.980]So that's just a further direction just to assess how, um,
[00:12:57.000]that liquid binding we, um, are able to just, uh,
[00:13:00.390]look how the radioactive tags are bound to the different receptors.
[00:13:05.760]And then next week we are just going to grow up some more clowns and kind of
[00:13:09.990]start the process all over again, starting from, um,
[00:13:14.820]I believe that we're going to start from the transformation.
[00:13:17.190]So with the Biel 21 cells,
[00:13:20.100]but that is basically all of my research this semester.
[00:13:23.790]So thank you. That's basically all I've done.
[00:13:26.520]Thank you for listening to what I have been doing all summer.

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Bacterial expression and ligand binding of purified domains of Stabilin-2

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