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  • ELIZABETH NOLAN: Today's recitation

  • will all stem from this reading by Youngman and Green

  • on ribosome purification.

  • But also, we posted an optional-- well,

  • not really optional but an additional reading

  • that's a review that talks about purifying macromolecular

  • machines from the source.

  • And I guess one thing I'd just like

  • to say about this review, something

  • I like is figure three because it indicates

  • many, many different types of methods that can be used

  • to purify some biomolecule.

  • And I think often it's easy just to think all the time,

  • well, let's just use a tag.

  • And tags have enabled many things,

  • but there's also many possibilities

  • out there and decades worth of work

  • before tags for how to purify proteins.

  • And in some instances, you might not want to use a tag

  • and that's discussed to some degree here.

  • So I guess I'm curious.

  • What did you all think about this week's paper?

  • Did you like it?

  • Did you not like it?

  • Was it easy to hard read?

  • Did you read the paper?

  • AUDIENCE: It wasn't too bad.

  • I've purified--

  • JOANNE STUBBE: You need to speak louder

  • because I can't hear you.

  • AUDIENCE: It wasn't too bad.

  • I've purified proteins before, so I

  • felt like I could follow along.

  • ELIZABETH NOLAN: So not too bad means

  • it was easy to understand and follow the text there.

  • Yeah, so compared to the reading for recitations two and three,

  • this one was probably easier to work with,

  • but there's a lot of details in this one too there.

  • One thing, I guess, I like this paper from the standpoint of it

  • being a methods paper, is the amount of detail

  • they give with how they did this purification and things that

  • didn't work, right?

  • So often, sharing those details with your readers

  • can make such difference for an experimentalist

  • in another lab in another part of the world, right?

  • And I think, from this, one with biochemistry training

  • could go into the lab and reproduce their purification

  • there.

  • So it's a good example in terms of what details to include

  • and what types of pitfalls to include.

  • So how many of you have done a protein purification,

  • either in a lab class or in your research?

  • OK.

  • And so what type of purification did you use?

  • AUDIENCE: We used a histamine-tag with nickel.

  • ELIZABETH NOLAN: OK.

  • So you used a His6-tag, probably a polyhistamine tag

  • in a nickel NTA column.

  • For everyone else, has it been that type of methodology

  • or another methodology?

  • AUDIENCE: I used the same one.

  • ELIZABETH NOLAN: OK.

  • OK.

  • So would someone like to kind of comment

  • on the basics of affinity tag purification?

  • So how does this work?

  • And why did you do it?

  • So what are the advantages?

  • AUDIENCE: So you have a light chain

  • of histamines on your protein, and you

  • use nickel, which is a metal that chelates

  • to histamine very well.

  • Is that right?

  • ELIZABETH NOLAN: Yeah.

  • The nickel's bound to something though.

  • You have the NTA ligand on the resin.

  • AUDIENCE: I did this over a year ago.

  • Sorry.

  • AUDIENCE: So if you have whatever

  • the type is, the [INAUDIBLE] bound to some solid substrate.

  • And then you can elute everything

  • that's not bond to that.

  • Elute what it does bind.

  • So you just tag the protein.

  • It's bound to a high concentration

  • of the free ligand.

  • ELIZABETH NOLAN: Or right, to push it off.

  • So the idea is that you have your biomolecule of interest,

  • whether that be a protein, which is what we're all

  • most familiar with, or the ribosome,

  • and then you attach a tag.

  • And that tag can be any number of things, right?

  • So in this paper, we saw they used a stem loop structure

  • incorporated into the 23s rRNA.

  • And the idea is that you're going

  • to use that tag to separate your biomolecule of interest

  • from the complexity of the cellular environment

  • there, so all of the other proteins.

  • And so you have some bead or resin

  • that this tag can bind to.

  • So in the case of the nickel column,

  • the His6-tag will bind to the nickel NTA on the resin.

  • And then you can wash away other components.

  • And then you devise some method to elute the protein

  • you hope to have trapped there.

  • So what are some advantages of using an affinity tag,

  • just thinking about this generally

  • before we delve into the paper?

  • AUDIENCE: Easy to install.

  • ELIZABETH NOLAN: OK.

  • So what do you mean by easy to install?

  • AUDIENCE: If you wanted to just encode

  • a 6 His-tag at the terminus of your target protein,

  • I don't know if you can say it's trivial.

  • It's easy.

  • ELIZABETH NOLAN: I'd agree it's easy.

  • There's many plasmids available that you

  • can insert your gene of interest into in order to have

  • this tag genetically encoded.

  • So when you express the protein, the tag's there.

  • So beyond that, from the standpoint of purification--

  • AUDIENCE: It's more specific.

  • ELIZABETH NOLAN: Pardon?

  • AUDIENCE: It's more specific.

  • ELIZABETH NOLAN: More specific--

  • AUDIENCE: Pure proteins as opposed to various charges.

  • AUDIENCE: It simplifies the purification.

  • Instead of doing size exclusion and ion exchange

  • that is much different.

  • ELIZABETH NOLAN: So the hope is it simplifies the purification

  • because you have some way initially

  • to pull your protein of interest out of your cell lysate there.

  • So that can be a big help.

  • What are some potential disadvantages of using a tag?

  • So have any of you run into trouble with a tag in the lab?

  • AUDIENCE: Having a tag can highly deform your protein

  • and change it.

  • ELIZABETH NOLAN: Yeah.

  • So it might change your protein or deform it.

  • What do you mean by "deform?"

  • AUDIENCE: It could just cause a conformation change

  • or the tag could make it localize somewhere else based

  • on size.

  • ELIZABETH NOLAN: Yeah.

  • So that's an example, say if you were doing a study in cells,

  • say, rather than a purification.

  • But maybe you tag a protein and it

  • goes somewhere other than it would go untagged right?

  • And that will affect your observations

  • and your data there.

  • And it might alter the conformation.

  • So it might affect the folding, right?

  • It might affect the oligomerization.

  • His-tags bind metal ions.

  • So is that a factor to consider there?

  • If you have an enzyme, will the tag affect activity, right?

  • And these things can be a positive or a negative.

  • Sometimes the tag is helpful in these regards.

  • You can't get soluble protein without the tag, right?

  • And sometimes the reverse.

  • You decide to express your protein or biomolecule

  • with a tag and you find out you get an aggregate, something

  • that the protein shouldn't be.

  • So these are just things to keep in mind when designing a fusion

  • protein and thinking about how you're

  • going to use an affinity tag