cc
I'm Tom Zinnen. I work here at the UW-Madison eveBiotechnology Center.ite I also work for UW-Extension
Cooperative Extension.
On behalf of our other sponsors, Wisconsin Public Television,
UW-Madison Science Alliance and PLATO, thanks again for coming Wisconsin Alumni Association,
night, 50 times a year. Tonight, I get to introduce tto you a very special person We do this every Wednesday
Dr. Richard Burgess, who was the for the Biotechnology Center.
founding director of the Biotech Center, and who was key in
building, which we entered back on October 16, 1995, not that it designing and building this
was a special day to me. Dr. Burgess is the
James C. Watson Emeritus Professor of Oncology
at the McArdle Lab of Cancer Research here at UW-Madison.
He founded the Biotech Center in 1984, and was its director until 1996. He was born near
Seattle, Washington,
on September 8, 1942. He obtained his bachelor's in chemistry at Cal Tech
in 1964, where he carried out undergraduate research
on Phase M13 with Robert L. Sinsheimer.
That is, he was doing that
research when he wasn't playing varsity basketball for Cal Tech. He obtained his PhD in
biochemistry and molecular
biology with James C. Watson at Harvard in 1969, elucidating the subunit structure
of E. coli, RNA polymerase, and discovering the first transcription factor, sigma.
Postdoctoral Fellow at the He was a Helen Hay Whitney
from 1969 to 1971. Institute for Molecular Biology with Alfred Tissieres in Geneva, Switzerland,
Then he came here to Madison to join the faculty of the
while on a Guggenheim Foundation Department of Oncology in 1971
Fellowship during the sabbatical year of '83 to '84, he was
biochemistry at Genetics Systems acting director of research in
Corporation back in Seattle. It was there where he learned how to make murine or mouse
monoclonal antibodies for use in his biochemical studies. He's published more than
biochemistry and molecular 230 research papers on the
biology of RNA polymerase, transcription factors,
on protein biotechnology and gene expression;
and nine master students. He was co-editor with on tMurray Deutscher of theery. He haMethods in Entomologyents,
Volume 463, entitled
"A Guide to Protein Purification, Second Edition," published in October, 2009.
Here at UW-Madison, Dr. Burgess teaches courses in
topics in biotechnology. protein purification, and characterization, and also
and he is the chair and has been He is Editor-in-Chief of the journal called Protein Expression and Purification,
instructor since 1992 of a two-week lab course on protein
two weeks each April at Cold Spring Harbor Laboratory on Long Island. He was cofounder of the
UW-Madison Center for Biology Education, which serves to
education of the public. promote and improve biotechnology and modern biology
from 1992 to 1995. He served on the National Research Councils Committee on Bio-based Industrial Products
He was the recipient of the 1982 Pfizer Award for Outstanding
Chemistry and the 1999 Contributions to Enzyme
Watson Medal for discovery of the first
He was awarded the United States transcription factor sigma.
Department of Agriculture Honor Award for work in developing bio pulping,
an environmentally friendly technology in June, 1997. In 2003, he was elected a Fellow
Microbiology, was elected in 2007 to the Council of the Wisconsin Academy of Sciences, of the American Academy of
elected as Vice-President for Science in 2011. Arts and Letters, and was
In 2008, he was elected a Fellow of the American Association for
the Advancement of Science, otherwise known AAAS. In 2001, he co-founded Conjugon,
Antimicrobial Biotech Company. a Madison, Wisconsin based
he has served on the Board of He cDirectors for thes on tWisconsin Biotechnologyf and Medical Devices Association,
which is now called Bioforward, from 2003 to 2006.
research into growing Wisconsin biotechnology businesses and He still plays an active role in translating basic university
corporations, and in educating the public about biotech.
In addition to playing varsity
basketball at Cal Tech, he still plays buckets three times a week.
His hobbies include serious winemaking, rock climbing,
and amateur archaeology, woodworking,
wild flower gardening and fly fishing for trout.
for which I'll be grateful On a personal note, Dick is the person who hired me,
for a long, long time. And also he's the person who
the outreach program and to the undergraduate lab course. These are really remarkable cresources for biotech outreacho
for welcoming people to this
campus. I think it's a great contribution to the
Wisconsin Idea, here in the year
of the Wisconsin Idea and I very much appreciate it. One of your parents was
a teacher?
>> Both. >> Both.
that's a tremendous commitment And that shows and I think
on the part of Dick and tremendous vision. Tonight, you get to hear about
One Second Suffices.
He's got a very interesting rapid oil and water method of purifying proteins.
It's a great pleasure to be able
Richard Burgess: Thank you, to welcomeTom.k Burgess to Wednesday Nite at the Lab. [applause]
When we hired Tom, we were trying very hard to increase
outreach to the general public
narrow almost. about science, particularly biology, because we thought biotech was maybe a little too
But he's done a wonderful job, and all these programs, like
to introduce to you what goes on this one, are his ways of trying
around here. Those of us who are on the
just a little over 40 years, faculty have been here
40 years and two months. We're used to seeing exciting things all the time.
And I admire your determination to come out on a lousy night and
and say that was interesting. Only my mother-in-law hopcalls me Richard.you somMy name is Dick Burgess.ome
I think you heard all the introductory stuff. I didn't know Tom was going to
I want to just right up front say that this work I'm talking read it all.
The real discovery came from about is actually-- I played a secondary role in it.
Dave Beebe's lab in biomedical engineering and from his
and graduate student post-doc Scott Berry
professor in oncology, Lindsey Strotman; and from my senior scientist, Nancy Thompson; and then another
get across is that these days it's hard to do research by studentyourself.ackson. SAnd collaboration among groupso
is common.
different language than Abiologists, although reallyy I was trained as a chemist, so I'm sort of in betweent
somewhere, but this kind of a project doesn't happen if you sit in your office and
just like you do. This is the kind of a project that happens when you start to talk to people who think
a little different. And so, I just turned 69. I thought I would be closing brainstorm with people who think
sunset about now. But as you'll find out, not only my lab and riding off into the
did we sort of get a new lease on my research career, but we
just recently got a grant from
the Gates Foundation to develop this technology to hopefully help find low-cost diagnostics
for developing countries. But what I want to tell you is sort of a story, a story about how our protein
work sort of blended into our interaction with
Dave Beebe's lab, and how that has dyear or so.er the last
So my passions are the what work I did my thesis work on
which makes copies of genes from DNA into RNA, but I'm in graduate school, which was the enzyme RNA polymerase,
my effort has gone into teaching not going to talk about that. In the last 20 years, a lot of
Insulin is a protein. aAll the things that are, aimportant are proteins, except proteins, enzymes or proteins.
about DNA. for maybe a little bit
But I love proteins. I do a lot of work related
about proteins. Another thing I really love is to proteins and teaching people
interdisciplinary research and brainstorming, as I said.
I really like to be stimulated
by people who think about similar, general problems, but from a completely different
perspective.
I also like to think about, occasionally think about how what I'm doing can be used.
The year I spent on sabbatical
in a biotech company, that was one of the first companies
kind of antibodies that started to make a special
called monoclonal antibodies. They were diagnosing diseases, thinking about treatments
for cancer. It was really exciting. I got to thinking, you know,
I'm a professor of oncology in the McArdle Laboratory for Cancer Research, but if I discovered something,
no one would ever be able to be treated by that discovery until there was a company involved in developing it,
and making a product and
going through the clinical trials that are needed. So I became very friendly to the
idea that scientists
need to really think about, is there a way I can use what I do to create products
that can help people? So I'm very interested
basic research results, and convert them into potentially commercial entities that can how do you take research,
I'm also very interested, as Tom said, about education. When I started being Director hof the Biotech Center in 1984,
everybody was afraid of biotechnology.
It was scary, because people didn't really understand.
Is there a problem...?
People were afraid of the science because they didn't understand it.
public lectures a year, just And I gave something like 40
trying to help people understand what biotechnology was. I've many times, had people come
up to me afterwards and say,
I came in here against what you're doing and now I think it's great, you know.
So it was a wonderful thing to
see them change their attitude once they learned a little. And to me that's an important
thing.
So I think letting-- We also, in a good society, base our public policy on facts.
And in fact,
a little bit of it, and that's for the public to use science to make good public policy, they need to understand
why I think it's impressive that you're all here trying to learn something about the science.
And finally I am very enthusiastic about what I do, and I just love to share my enthusiasm with people who want
to hear it. So that's some of my passions.
in the audience will know a lot of what we're talking about, and So I was told by a professor of mine one time that some people
some people won't know anything. If you talk to the few who know
a lot, you're going to lose everybody else. But if you talk to the people who don't know very much about
it, you're going to,
it's going to be a little boring to the people who know more.
people as much as you can as quickly as you can, so that So the strategy is to teach
what you're saying. So I'm going to try to do that. youI really don't know yourge where they can follow
feeling that you have active backgrounds, but I have a
minds and that you are interested in learning. So the first part of my talk
is really, it's a short part, but it's about the work my lab does on making monoclonal
in protein purification. antibodies and their use
So who knows what a monoclonal antibody is?
Okay. Few people. Have you heard of an antibody? Who's ever heard of it?
So if you inject a rabbit or Okay.
mouse with a protein, it will make an immune response. That's what happens when you get
vaccinated against the flu. You're making an immune response. You're making antibodies against
the flu virus.
Well, you can immunize a rabbit or a mouse against a protein that you're interested in,
and it will mount an immune response, and make antibodies,
it will react with that. The thing you inject,
the vaccine, is the antigen.
That's the thing that causes the animal to make the antibody.
molecule that can specifically The antibody is a protein
bind to the additive. Monoclonal antibodies were a major development that occurred,
I don't know, in the early '80s,
or late '70s, which allowed, it's just a better way
You can make lots of them. to make antibodies.
You can make very specific homogeneous antibodies, but it's
We found a way of using what we call gentle release antibodies. This little thing is what I call really immaterial for this talk.
We found a way to make monoclonal antibody.
antibodies that would bind onto a protein, but then release it easily.
And we call these things
polyol responsive, because they respond to a polyol, like propylene glycol, a couple
carbons with a O-Hs on it.
That's what a polyol is. Glycerol is a polyol. But, again, it's really not
important, except to say that this class of antibodies can
bind tightly, but it can release under gentle conditions.
If you have an antibody, a
monoclonal antibody that reacts with a protein, it doesn't react just any old place,
it reacts to one specific place.
That's called the epitope, that site that the antibody bonds to.
So if I know that this antibody binds to this epitope,
I can take that little piece of protein and I can hook it
on to another protein, and now
That's called an epitope tag. Finally, there's a thing called otgreen fluorescent protein.s that little epitope on it.
Jellyfish have this protein that You might have heard of it.
allows them to be fluorescent. A Nobel Prize was given a few years ago for the people who
discovered this, because it's been used widely in research. And I'll show you how we use it.
hesitate to ask, because if you have a question, I'm sure other people have a question. IfBut I will try to define myn't
terms.
Okay, so there's something called Immunoaffinity Chromatography.
This is where you use the specific binding of an antibody to pull out of a mixture
has the epitope. of proteins, the protein that
So in a cell, there may be 10,000 or 100,000 different proteins, but only one of them
has this little site that your
antibody that you've got will bind to. So you can actually purify that
protein out of a whole mixture
of proteins. That's called immunoaffinity. Basically, you have a little
bead that has antibodies. And antibodies are sometimes
represented as a little Y. Each end of the Y is a site that
will bind to an epitope.
Okay, so here's a mixture of proteins. So you put them on.
Only the yellow protein, which
is recognized by the antibody on the column, binds to the column.
through. Everything else flows right
So you add it, then you wash it, and you wash off some proteins
by accident, that might have been stuck
or even just trapped somehow. But you wash it. And then finally,
you elute it off with something that will weaken the interaction
So we use the antibody between the protein and the antibody.
in an extract. as a very specific tool to grab a very small subset of the total proteins
That's called "immuno," from antibody; "affinity," which means it has
an affinity or a binding
specificity for it; "chromatography," means that we've used a column to separate
our protein from everything else. So that's what immunoaffinity chromatography is.
Okay. This is green fluorescent protein.
structure. It's a beautiful, beautiful
It's called a beta barrel. It just has amino acid, a polypeptide chain
that wraps around and makes this beautiful barrel.
And inside there's a little part of the protein can actually
become fluorescent. So when a certain wavelength of light hits it, it will then give
that fluorescence is what you see. off a wavelength of light and
It's called red fluorescent protein. And it happens to be green. There's another related protein.
Guess what color it gives off? We took the epitope, the little
spot on our original protein
that our antibody bound to, and we grafted it on to GFP. So we had a GFP
that was epitope tagged.
So we can do that. You can make the gene for the GFP, and you can make
the gene encode for this little stretch of 15 amino acids.
tag of amino acids. Okay, and this GFP will bind Now you will make that protein. It will have that extra little
It's just a name we gave it when we made it. to this antibody, which we call 8RB13.
It's not a very catchy name, but that's what it is.
This is work that was done
in my lab. The first author was an undergraduate, a student helper
who came in and loved to do research and we gave her this project, and ended up writing a beautiful paper about it
and she's the first author. So one of the beauties of being an undergrad at a research
have the opportunity to work in a lab and actually do something. university like Madison is you
but this antibody, this kind of antibody I mentioned before, Okay, so I don't really want to go into too much detail here,
it's called a polyol responsive. It binds tightly to the epitope
and in this particular assay, called an ELISA, you get
a strong signal if it's bound. If it starts to come off,
the signal goes down. I really don't want to spend the
because it's not super important time it takes to describe all the details of this assay base
except to say that by increasing the propylene glycol, and increasing the salt, we can go
from a condition where the
interaction between the antibody and the epitope is strong and it stays that way,
to a situation where it comes right off.
So we can affect how tightly something binds by just changing
the salt. And that's actually pretty cool.
I mean, even at this 40% propylene glycol,
when there's no salt around,
it binds tightly. But when we start adding salt, it binds weaker and weaker.
Okay, and so, here's the visual.
beads, which have our antibody So here's a column. It's about this long. It holds two milliliters of
on it. So that's an immunoaffinity column.
We put an extract on
the beads binds to the epitope which had this epitope tag GFP. So it binds to the column because the antibody that's on
that's on the GFP. And it stays there. You can wash it, and wash it,
and wash it.
it doesn't come off. It never comes off. If you wash it with no polyol and high salt,
If you wash it with high propylene glycol and no salt
But if you take a combination of salt and polyol, then it comes right off. it doesn't come off.
There's one here, one here, one here. If you start putting on this So these are one mil fractions.
that will cause the protein to buffer, which is a buffer
release from the antibody, you'll see, about after two mils, you see your protein
come off. As that buffer runs through this zone, this fluorescence
and it comes off. And, again, this is an moves right down the column
experiment to show that by going down from half molar
to quarter molar
to eighth molar to sixteenth molar salt, what happens is it takes longer
for that protein to come off, meaning it's actually still binding a little bit, and it slowed down as it moves
down the column.
how tightly it binds. The uIf we add this buffer,this is an antibody which can bind epitope tag GFP, but we can vary
it's bound weakly. it comes right off. If we add this buffer,
So this is where we were If we add one of these buffers, it doesn't come off at all. It binds very tightly.
about a year and a half ago. We had been working on this.
Dave Beebe, who's a professor, And then I got a phone call from
as I said, in biomedical engineering, and they had developed a technology.
Since then, they've published a couple papers on this using it to purify RNA
Dave knew me because and to purify cells.
he had taken my course at Cold Spring Harbor.
Even though he was a professor, he came to this course So he was my student.
for two weeks, 14-hour-a-day lab course.
He knew I loved to purify proteins. And he said, would you be interested in helping us
demonstrate that our new
technology works with proteins? So I'll tell you about the technology.
And this is really what the interesting story is. But I had to give you a little
antibodies. So this was in May of 2010. We had this thing, bit of background about the
We're going to write a grant which we now call IFAST.
to the Gates Foundation to try to get some money to do research to develop our
technology into a diagnostic, so that we can treat people in Africa under a tree. I mean, a very primitive clinic
with no electricity,
no equipment, no refrigeration. And we thought this technology might actually be able to be
used that way.
So we did some experiments and it turned out we got great
and we got it. results and we put in the grant
$3 million grant. I think it's $3 million. $2 million, or something.
A lot of money. It was enough to help me support
will stay open for three years. And it just turned out that my this antibody, 8RB13 andee years, which means that my lab
as a model system for studying this epitope tag GFP was perfect
purification of proteins. Okay, that's the background. Okay, so a few more concepts.
Epitope tag GFP, I've already talked to you about.
It's a GFP molecule, a green fluorescent protein
that has this amino acid tag
that our antibody binds to. Affinity reagent is actually the antibody.
It's the thing that has some
affinity, in this case, for the tag. The paramagnetic particle
is sometimes also called the
magnetic bead, teeny little bead that if you put a magnet next to
through solution and stick to the side. A paramagnetic bead that has a tube, the beads will all move
that is coated with a covalently a protein G on it, is a bead
attached protein that binds antibodies.
the protein G bead with our monoclonal antibody, and now the So what we do is we mix
antibody binds to the protein T, and now we have an affinity bead
that is specific for binding the
epitope, in this case the GFP. An analyte in the parlance of diagnostics is the thing you're
trying to measure.
You know, you have a blood sample, you want to know whether
People who have malaria have a the person has malaria.
you're in a position to cedetermine whether it's there. If you can grab that protein, separate it away from blood,
and how much is there. And that's the concept of a
diagnostic, is to actually
measure, determine whether a certain thing is present. And finally an immiscible phase
in this case is some oil. Remember, oil and water don't mix.
of this technology. The term IFAST, which is the name we've given Well,to this technology isnciple
Immiscible Filtration Assisted by Surface Tension.
It's pretty obscure. IFAST is easy to remember,
but we'll talk about it some more.
So the basic idea is the following.
This is a device. This device, there are five of them on this.
glass slide with sort of It's basically a thin,
a silicone plastic with little wells, three little wells in it.
So you put a buffer over here. And they're interconnected.
an extract or whatever it is That's called the output. And over here you put a sample, like blood or a cell,
that you're starting with. That's the input. In between, you put a droplet
of olive oil. Now, you don't put
But the olive oil, balsamic vinegar in this one! [laughter]
In other words, they don't it can be other kinds of oil. Anything that is immiscible with water.
because this is so teeny, disssurface tensionher. holds the interface flat. Now, normally if you pour oil
on water, it floats, because
it's so small, the oil it's lighter than water, right? But if you put it into this little thing, because
that Dave Beebe is-- doesn't float on the water. It just stays right next to it. And one of the things
In my opinion, his strength is that he thinks small. I mean, he thinks about micro
channels and very small volumes.
And this is a consequence of thinking small, is that you can actually design something
where the oil will stay in this middle well and not mix with the water buffer on either side.
So you put your crude sample
in this side. You put in some magnetic beads that have an antibody.
Then you take a little magnet,
a teeny little magnet, and you just drag it
and you pull the magnetic beads that were here across like that
Now, that's just, you know, it's not rocket science. through the oil and over to this other side.
I mean, this is just pulling some little magnetic beads
through a little droplet of oil.
These are eight microliters. A typical drop out of an
So we're talking about something eye dropper is 50 microliters.
that's less than a sixth of a volume of a drop.
a sample, an oil phase, and then an output phase. And by this simple process But, anyway, so we have
and beads across into here, we've purified of pulling the magnet
whatever that antibody bound to. So this is, you know,
if the blue is the on-put or the crude sample,
the white in between is the oil, and the pink is the output,
then this is five devices.
Here's a configuration. It's easy to manufacture these with different shapes and stuff.
Here's one with a sample, oil,
They're a way of taking a crude sample of a magnetic bead carrying an affinity reagent, Thletting it bind its analyte,.
pulling it through the oil
to the other side. So in that one second or so that it takes
to drag those beads across, you've purified your sample.
I mean, and I have to say, I So this is pretty astounding, in my opinion.
just sort of step back a minute. Scientists live for the time
where you now have when something happens
it was a very exciting time awhen we got these resultse that I'm going to show you now And, you know, it was just,
of all kinds of ways because we could just think
you might be able to use this simple technology
through a droplet of olive oil, something as simple as that, of drawing magnetic beads
and yet this is something that people really hadn't done before
and in a way that allowed the kind of flexibility that
we have with multiple wells, and so on.
So this process again is called Immiscible Filtration Assisted by Surface Tension It really means that the oil
stays separate from the water and you can draw the beads
right through, from the water, from the aqueous phase
through the oil into the other side.
So here is a little video of that process. I want to just show you a little movie.
is the magnet. So let's see, if I can go to... This thing you see right here
This magnet. And when you put down on it the little device,
and this has several, you see the dark things?
It pulls them through. Those are the magnetic beads. You pull it.
Now it goes into the oil and comes out the other side. That's it.
I'll do it again. [laughs]
So there's the little-- the on-put well with the beads. Having bound something, now you pull it through.
Now it's in the oil.
And then it's back into the output well. Now, it turns out that when--
There's lots of little beads
there. When they go through the oil, they sort of ball up
into a blob.
It turns out that the liquid on the outside of the blob
right off by the oil. And so you carry through is just sort of squeegeed
very little of trapped liquid. As a result, this is very
effective.
For those of you who do experiments in the lab,
as effective as three washes. So normally you take, I mean, the traditional method, if you this turns out to be
you take your magnetic beads and suppose this is a little tube,
you mix them with your sample, just like we mixed our beads
on-put well. with our sample in that
Then you hold the magnet to one side.
Then you suck away the liquid, The beads move over.
You do like this because you've removed whatever you want. You put some buffer in.
The beads all move over for ato one side.tes. Then you take the magnet, you put it like this.
You put some more buffer in. You suck off the liquid that doesn't contain the beads.
You mix it up. You do that three times.
washing is equivalent to passing those beads It turns out that all of that
part about this, in a way, through a droplet of olive oil. And that is the really important
because it really means that in a one second, you can accomplish what normally takes ten minutes.
Now, that's cool, but, you know, in itself--
It turns out we found a reason why that was much cooler
than we realized at first.
Okay, so we've got And I'll try to explain that. So let me go back to my other thing here.
our little setup here. So I'm going to go through this fast because I really introduced
a lot of this.
The device itself is this silicone type plastic. There's an on-put, an oil phase,
and an output chamber that
are about eight microliters. We have a little magnet.
We have oil, which can be olive oil or it can be We have a buffer.
some other thing like a fluorocarbon oil.
We have a paramagnetic bead to which we can bind
a monoclonal antibody. In this case our analyte is the
green fluorescent protein with the epitope tag on it. So we can actually see this happening.
The contaminant, you know,
what you want to do, in this kind of an assay, you want to see if it's selective,
so a key term is selective, meaning it grabs what you want, but it doesn't take anything else.
And you want it to be efficient.
You want it to grab everything that's there and pull it across.
of your analyte that's captured and pulled across and purified. And you don't want to pull So you want a high yield
So those are the two things that are important. So we took red fluorescent anything else.
but it doesn't have the epitope tag on it. protein, which is red,
It shouldn't bind to our bead, because it doesn't have
the epitope that the antibody can grab onto.
We mixed it in with this broken E. coli,
just a cell lysate, a mass of proteins, and we did the experiment.
Now, here's a schematic. So, here's the beginning.
The beads are in the input well with the crude lysate. There's green fluorescent
protein.
You can photograph this under a light that will show the green fluorescence, and you
can photograph it under a light that shows red fluorescence.
So these are two different pictures.
So they're both over here.
We move it across. Now the green fluorescent protein is over here,
but the red fluorescent protein stays behind. It turns out that something like less than 1%,
of the red fluorescent protein
gets carried across, and it turns out that something like 95% of the GFP
is captured and pulled across.
It turns out that the blue here is conventional, three, you know, magnet
on the side of a tube with three washes that take about ten minutes,
where we basically took one second. So you can see that the results and theare very similarT thing
between the red and the green.
This is how much GFP is left behind, and this is how much GFP
is pulled across; and this is how much RFP
is pulled across. So very similar property, performance, only instead of and this is how much RFP
we've washed for one second. washing for ten minutes,
So that was the thing. This is a log scale.
not even on a protein, and we showed that there's this amount if you do one, two, three, So we put some fluorescent dye,
You're down to like .2% of this. or four conventional washes.
So one passage through If you do one or two passages through the oil phase, you're down even lower than four.
is between two and three washes, and two passages
So very effective washing, is better than four washes.
very simply. Just pull the beads from here,
through a little wash and on through the oil through oil,
to your final destination. So we were saying,
this is great, this is great. We can write a paper. We can show that this works.
Then we got to thinking, wow, you know, this is so much faster We can purify proteins.
more to this that we can take advantage of. So this is a little sidetrack this, for a minute here.thing
If you have two things, A and B,
they're bound is something and they bind to form AB, they also can dissociate. So the measure of how tightly
called Equilibrium Dissociation Constant, or Kd. Even I don't understand this
as well as I should, so I'm not going to try to explain it, except to say if this is
it means that it's very tight. a very small number,
will dissociate with a half-life So ten to the minus ninth is a very small number, and things that bind that tightly
of about 30 minutes. That means that if I have pure AB, and I put it into
a tube, about half of it
will dissociate it into A plus B in 30 minutes.
If it's 1,000 times weaker, then its half-life It stays together really well.
put it in and you wash it, is two seconds. That means, that if you
and you hook one of these on, you know, onto an antibody on a column,
and you start to wash it, in two seconds, half of B has fallen off. In another two seconds
another half has fallen off.
In another two seconds another half as fallen off. And so in a minute
there's nothing left. And so that's exactly-- What we realized was that even if something
had a very short half-life, let's say ten seconds,
there's no time for B to fall off of A. And therefore we can capture second to separate it away,
You may not fully appreciate very weak interactions.
why that's so important, but I'll try to explain.
Nobody appreciated this a year ago. This is a relatively new Because I didn't appreciate it.
It turns out that many things in the cell are really concept.
the enzymes that make DNA, multi sub-unit complexes, the enzymes that make RNA,
proteins from messenger RNA. the enzymes that repair DNA damage. The ribosomes that make
Almost everything you can think about that goes on in the cell is carried out by a machine
made of multiple proteins.
Now, when we purify one of those machines, we go through many steps of purification,
many times where it's bound to a column where we wash it
for a long time. So we're only isolating those
sub units that hang together
a pitcher of beer that there are proteins that are very important parts and of those complexes, you
which we've never seen, because when we grab onto that complex
and wash it, we wash off all those things
that dissociate with a half-life
we see what's tightly bound, of two seconds. We lose them. And if we look at what's left,
but we don't see the things that were weakly bound and came off. So we think that we have a
method for isolating complexes
that nobody's ever been able to see before.
It's starting to become apparent These turn out to be--
that this kind of interaction may play a very important role in biology.
It's just that it's been
impossible to study. So here's a this simple thing,
through a droplet of olive oil, and all of a sudden you have a new way to study drathe biochemistryads
of cellular processes.
That's what makes one get up in the morning and go to work,
like that will happen, and is the chance that something
you will all of a sudden say, this is really neat, and think of all the ways
we can use it.
After I gave a talk to my department about a year ago, and there's 22 faculty
in my department other than me, 20 of them came up to me
I need to do experiments where right after my talk and said, I've got to have this.
I can study weak interactions. How do I get these things?
I've gotten by all scientists, So, anyway, that's the response
by chemical scientists, that I've talked to. When they hear about this,
they immediately think of ways
just more of an illustration. tHere's the bead. for their own research. So here's an example,
There's an antibody. It's binding
the yellow molecule.
But there is another weakly-bound red molecule.
the red molecule falls off When you wash it,
until when you finally elute the yellow off the antibody,
It's dissociated. Where with our case, we have there's very little red left.
involved, we can-- some red bound to the yellow, but since there are no washes
when we elute off the yellow, along comes the red. So we can identify that.
There are ways of determining what that protein is. That's a whole other story
So it has the potential to see things that using mass spectroscopy.
NIH grant that we just put in. were previously unseen. That's what we titled our
It's called "Seeing the Unseen." It sounds a little mysterious.
exciting to realize that-- But to me, it's incredibly
It turns out that in biology, there's lots of things that were there
and nobody ever saw them, because they didn't have
the right tools. The history of any science
is that way.
you see it and you understand Bthat, you know, you've mades alonjust a huge leap forwardect something, now all of a sudden
when you can start to better in understanding how things work
understand what are the critical components of this complex.
I'm not sure, this may be rubbing it in, but again, remember our antibody that was sensitive to salt?
Very low salt,
it bound very tightly, and at very high salt,
Well, here we took the it bound very weakly?
epitope tag GFP and we pulled it through with the antibody bead
What we found was that our IFAST at different salts.
method captured over 90% of this stuff.
where the binding was getting pretty weak, it came down. When you got to the salt
So if it's too weak, there's nothing bound to pull across.
Using the conventional method,
much less sensitive right off the bat. So if you go into this range
right here where this protein is dissociating quickly,
yet we see it extremely well you can hardly see it with the conventional method,
with our method. We've used this now, there must be a half a dozen or more
examples of where we've identified weak interactions
that have been extremely difficult to detect before.
But these are known interactions. But we can see them, where other
What we're now wanting to do people could barely see them.
is look for things that nobody's even barely seen. So the conclusions are
that this IFAST technology, this basically fabricating
pulling an affinity bead a little linear device that involves oil and water phases,
through the oil to wash, has very good yield.
Very good selectivity. We've gotten 95% recovery.
A single phase is as good as We can discriminate against the things that shouldn't be bound. And it has extraordinary speed.
two to three conventional washes and we can use a double phase device that gives us
better than four washes.
We didn't even know that this technology existed. But when Dave came to me not with this in mind.
show that our technology and said we want you to help us
will work to purify proteins, we had the perfect system ready. It was just one of those
serendipity things, where you just have the right thing in the right place
things happened very quickly. We can therefore isolate at the right time and
these weakly-interacting proteins in amounts
that are quite detectable.
We can see them on gels. We can see them on something
which is a very sensitive immunological detection. called a western blot,
which are the three highly I'm sure there's plenty there to see in a mass spectroscopy,
sensitive ways that you can look for things that were bound. So we're now trying to develop
and show that it works well with mass spec, and it can be used to detect
important proteins, and that's unknown, but physiologically
the basis of the NIH grant that we're working on.
other people have used it to find things that were weak, We've used this, and
and now we can see them. And coming back
to the Gates' grant,
we are working with Dave's lab on what I would call
low cost protein isolation an extremely simple,
that's part of a point of care diagnostics system
So what does that mean? Well, if you're sick and for use in developing countries.
you live in a little village in Africa somewhere,
and you walk a day and a half to a clinic and they say, well, you're clearly sick, but we're
but let's take a blood sample and we'll send it in not quite sure what you have,
to Johannesburg. And in a month we'll get
the results back, and then
you come back to the clinic and then we'll be able to treat it, maybe.
Well, the person
may not come back. Or they may have died in the meantime.
I mean, what you'd really like
We're going to put antibodies tthat will bind to antigens okay, take this blood sample, okay, put it into these well.
We're going to do this, and then associated with various viruses and pathogens.
we're going to determine how much of those, whether those
blood sample, or that urine sample, or that sputum sample. pathogens were present in that
Then we're going to, right away, know what you have, and right
now, we're going to give you the
treatment. So it's point of care.
place where you're going to be That means that right at the
giving the treatment, you diagnose what it is. There's no lag for a month while
somebody, the patient proceeds
the tree next to the river has to get worse and worse. But the challenges are that I guess a clinic underneath
But it has no $500,000 pieces of no electricity, or very little. Maybe it has a solar panel, maybe not.
equipment that a normal hospital might have for diagnosing,
It has no refrigeration. for doing blood chemistry.
All this stuff has to be able to withstand temperatures that get up into the 100s, 110s, 120s,
high humidity, not ideal
conditions for storing biological materials. Anything that you would come up
with would have to be simple. It would have to be cheap,
device if it's going to be used in that situation. I mean inexpensive. I mean, it can't cost $25 per
It's got to cost and be much cheaper than that.
You have to, never the less,
be accurate. It's got to be able to tell you,
And it has to be robust. is that antigen there or not.
It can't require that you deliver your blood droplet highly precisely.
You just take a little
it shouldn't matter. thing you put in, whether you put six microliters, or eight microliters, or 10 microliters,
It should be insensitive to that kind of variation, because you don't
expect equipment to be available
to deliver things that precisely.
It's a real challenge, So it's a challenge.
but the idea would be, and so what the Gates Foundation
we're going to give some grants for people who are collecting samples. did was to say, okay,
You have sputum. What do you?
It's viscous. Do you add an enzyme that will break down the viscosity so that
the sample is easier to handle? How do you take and pre-treat the sample? How do you do this?
This is all part of the equation. How do you analyze
If its nucleic acid, you can do something called a polymerize chain reaction, which amplifies what cit.s out?
But how are we going to sensitively determine whether there's a malaria antigen there or not?
Then finally, after you've done that, you've got to integrate those results in a way that can be downloaded, sent to a cell
phone, to someplace they can be stored and where the person at
would be the appropriate the clinic can use that information to decide on what
treatment. It turns out that the Gates Foundation funded 22 grants
that cover these various areas. The Grant Council in Canada has had a similar project, and
So they've actually got another one here, which is in communication. we decided to join forces.
the ideal thing, it's not clear that it would actually be widely It turns out, even if we had
disseminated and used. Most of these countries don't
have any policy for certifying
diagnostics. It's sort of sad. So there's a whole political
thing here, which is how do you get people, even if you have
How do you get the physicians to trust it? the ideal thing, how do you get people to use it widely?
particularly true there. This is true of all new diagnostics, but it might be
But the impact could be very large.
that starting out, thinking about how do we purify proteins in a very abstract way, that It's sort of exciting to think
maybe we can provide an important component that might we've led to a situation where
places that do not have the expensive facilities needed that allow rapid diagnostics and treatment of disease, even in
in Dave Beebe's lab, recently a company has been started we have in a modern hospital. So this device that was invented
This is to answer the question called Elaion, which is the Greek word for oil.
to be making these things. when my colleagues say, well, how can I get this. Well, this company is going
Bill Checovich is sitting in the third row there.
most of the business development He's the person who's been doing
work for Elaion. I think he's been getting a very
both scientists and companies who want to start to use this. positive response as he talks to
We have the market here, potentially in diagnostics, but
there's also clearly a market in the research arena,
in the drug development arena. So it's maybe the
pharmaceutical companies who
will want to use this. We'll see.
[applause]