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

Lauren Vatter Author

04/05/2021 Added

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This presentation describes the research being done on the Stabilin-2 receptors.

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[00:00:00.740]Hi, my name's Lauren Vatter
[00:00:02.290]and I'm going to be talking about
[00:00:03.880]the bacterial expression and ligand binding
[00:00:06.180]of purified domains of Stabilin-2.
[00:00:08.952]So just a little background introduction to this project,
[00:00:13.390]heparin and antisense oligonucleotides
[00:00:16.350]which is shortened to ASOs
[00:00:18.000]are both catabolized by Stabilin receptors.
[00:00:20.650]So heparin is used in a lot of medicines in modern society
[00:00:25.010]and antisense oligonucleotides, ASOS,
[00:00:29.180]are using a lot of gene therapies.
[00:00:32.950]So they're both pretty common in the medical field
[00:00:36.300]and catabolized by the Stabilin receptors in our bodies.
[00:00:41.040]So this is mostly done by Stabilin-2
[00:00:43.040]and to a lesser extent, Stabilin-1,
[00:00:45.410]but in this project, we only focused on Stabilin-2
[00:00:48.940]and there were two different Stabilin-2 receptors.
[00:00:52.890]So there's one that's 315 kilodaltons,
[00:00:56.830]that's what we call Stabilin-2,
[00:00:59.340]and then there's one that's 190 kilodaltons
[00:01:02.500]and that's 190-HARE.
[00:01:04.890]And 190-HARE and Stabilin-2 have the same affinity
[00:01:08.840]for binding ligands
[00:01:10.150]so that indicates that all of the binding happens
[00:01:14.360]on the latter two thirds of the receptors
[00:01:17.810]since 190-HARE is shorter than Stabilin-2.
[00:01:23.320]And so out of the 19 ligands
[00:01:25.970]that we know bind to Stabilin-2,
[00:01:29.840]only two of them have been characterized
[00:01:31.690]in terms of binding.
[00:01:33.130]So the objective of this project
[00:01:35.950]is to discover how the other 17 ligands bind to Stabilin-2
[00:01:40.030]and the research question
[00:01:42.750]that kind of guides this process is,
[00:01:45.900]what region on Stabilin-2
[00:01:47.440]interacts with electronegative polymers?
[00:01:50.630]And so there is a stepwise process
[00:01:53.850]in which we use to discover what and how binding works
[00:02:00.110]on Stabilin-2 and that starts with PCR.
[00:02:03.120]This whole process is kind of outlined in this graphic here,
[00:02:07.730]off to the side and so we start with PCR.
[00:02:11.700]So we use primers that anneal
[00:02:14.070]to the third and fourth EGF domains,
[00:02:17.660]known as Z3 and Z4, respectively, on 190-HARE.
[00:02:21.500]And using these primaries, we amplify them by PCR
[00:02:27.007]and we ligate them into a suitable vector.
[00:02:30.120]So the vector that I work with is pET-43.1a.
[00:02:35.000]And by ligating them into this vector,
[00:02:37.750]the protein, the recombinant protein
[00:02:40.690]will have a success His-tag
[00:02:42.880]which is important for the protein purification
[00:02:46.300]that I'll mention later on,
[00:02:48.190]and after these plasmids are transformed,
[00:02:51.500]or after the Z3 and Z4 has gone through
[00:02:55.950]and ligated in the pET-43.1,
[00:03:00.110]the new plasmids are transformed into E.coli
[00:03:04.970]and then we have to verify
[00:03:07.391]that actually the plasmid's in there
[00:03:09.381]and the transformation went correctly
[00:03:12.060]so we do that through PCR or shift of enzyme digest
[00:03:17.500]to verify the size of the plasmid
[00:03:19.860]and make sure that there's no frameshift
[00:03:23.650]or deletions or any other mutations.
[00:03:26.840]And then after the protein on the plasmid is verified
[00:03:30.650]then protein expression can begin
[00:03:32.820]and so this is when the plasmid will be transformed
[00:03:38.200]into the BL21 Origami E.coli
[00:03:41.670]and it's transformed to this specific strain of E.coli
[00:03:45.210]because it is specialized for folding
[00:03:47.760]which is really important
[00:03:49.190]for the protein expression of Stabilin.
[00:03:53.200]And so the next step is protein purification
[00:03:56.150]and so after the protein expression's done,
[00:04:03.120]we move on to protein purification
[00:04:05.220]and so it's analyzed in an SDS PAGE gel
[00:04:08.223]and the cells are fractured by French press or a sonication
[00:04:14.640]and so the recombinant protein and the cell lysate
[00:04:18.657]and the whole cells will all be analyzed in that
[00:04:21.150]and then the recombinant protein will be purified
[00:04:24.972]from the cell lysate using nickel chelate chromatography
[00:04:28.700]and so that's when the success His-tag's actually used
[00:04:33.430]because the recombinant protein will have that
[00:04:36.100]but the cell lysate won't, so that's where they separate.
[00:04:40.590]And then finally we have ligand binding.
[00:04:43.690]So when we purify the proteins,
[00:04:46.264]the protein can either be alluded through the column,
[00:04:49.384]through the column of chromatography,
[00:04:51.560]or it can just stay in the column
[00:04:54.050]and so the different types of ligand binding that we do
[00:04:58.000]will depend on the different techniques that we do
[00:05:02.020]for the ligand binding will depend on what happens
[00:05:05.440]with the protein, if we leave it in the column
[00:05:07.568]or if we allude it
[00:05:10.160]and so the data and results for this project,
[00:05:13.660]I am kind of an interesting special case
[00:05:15.880]when it comes to UCARE,
[00:05:17.690]most of the other people that are involved in UCARE
[00:05:20.530]have been doing this for basically the entire academic year
[00:05:25.970]whereas I only started with UCARE about two months ago,
[00:05:29.050]there was a situation where someone had a drop out
[00:05:32.530]and luckily enough, I got to be slotted in as a replacement
[00:05:36.170]but that only happened about two months ago
[00:05:38.140]and I think everyone can agree that
[00:05:39.760]that's not an ideal time period
[00:05:43.040]for getting concrete good data for research,
[00:05:46.550]it normally takes longer than that.
[00:05:48.150]So I haven't gotten any real concrete data
[00:05:51.190]from this project yet,
[00:05:52.360]but as you can see on the slide here, here
[00:05:55.740]are some gels that I have made.
[00:05:57.970]There's been several successful PCR products
[00:06:00.630]and transformations.
[00:06:01.990]These gels here are with the pET-43 plasmid.
[00:06:07.470]You can see the,
[00:06:10.050]it was just verifying that the pET-43 plasma
[00:06:13.720]actually had the recombinant protein in it.
[00:06:18.320]Further directions for this project are very exciting.
[00:06:23.640]So just last week we got primers
[00:06:25.870]for positions 6678 and 6745 on Z4.
[00:06:30.452]And so we'll be using those for PCR
[00:06:34.010]and then analyzing those with the recombinant protein.
[00:06:39.790]And so one and two liter cultures started growing last week
[00:06:44.330]and they're going to be used
[00:06:46.430]and assessed for protein expression this week.
[00:06:49.910]So we're just continuing trying to find
[00:06:54.300]how these ligands bind to the Stablin-2
[00:06:56.850]using different sections of the Z3 and Z4 EGF domains.
[00:07:04.800]And so finally,
[00:07:05.930]I would just like a couple of acknowledgements.
[00:07:08.270]I would just like to thank Dr. Harris
[00:07:10.743]for all the time and effort he's put in
[00:07:11.790]and allowing me to be slotted into UCARE the way I was.
[00:07:16.600]And then all the other undergraduates
[00:07:18.370]that work in the lab with me for this project
[00:07:23.370]and just on their own projects.
[00:07:25.430]Everyone works so hard in the lab.
[00:07:27.776]So I would like to thank everyone for that.
[00:07:29.230]So that's about all that I have for you.
[00:07:31.320]Thank you.

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

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