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NATHANIEL: Hi.
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My name's Nathaniel.
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As a scientist at MIT, I study how ultraviolet light damages the proteins
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in the human lens.
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And for my experiments, I purify human lens proteins from bacterial E. coli
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cells designed specifically to express human proteins.
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In this demonstration video, I'm going to show you how biochemists use column
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chromatography to purify a variety of proteins.
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In specific, today we'll be purifying GFP, green fluorescent protein.
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In lecture, Professor Landers talked about the incredible diversity of
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structure and functioning that proteins have.
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Protein purification is a critical step to understand that
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structure and function.
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By the end of this video, you'll be able to describe how biochemists use
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column chromatography to purify any number of proteins.
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Before we get started, let's talk about we'll be doing today.
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How do we purify one specific protein from all the other
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proteins in the cell.
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As Professor Landers discussed, scientists to start by breaking open,
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or lysing cells to release all the proteins.
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Once they have this mixture of proteins, how do they isolate one
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specific protein from all the others?
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Scientists can use a technique called column chromatography to take
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advantage of the different properties of their specific protein of interest,
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like size and charge.
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Some proteins are larger than others.
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Some proteins are more hydrophilic or hydrophobic than others.
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We can separate proteins by flowing a protein mixture through a matrix of
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beads in a column.
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The scientists select the beads for the column and the binding buffer
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based on the properties of the protein of interest.
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The protein of interest binds to the beads based on its affinity for the
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beads in the column.
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To remove other unwanted bound proteins that are more weakly
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associated with the column, scientists can wash the column with a different
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buffer that pushes the weakly bound proteins off.
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When removing the protein from the column, we use a third buffer, and
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collect fractions of liquid coming off the column.
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For most proteins, scientists don't know which fraction contains the
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protein of interest.
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They have to test each fraction for a specific activity.
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For today, we'll take advantage of the unique fluorescent property of GFP,
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and use its green glow to track the protein throughout our
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purification steps.
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Do you know that GFP originally came from jellyfish?
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GFP is a really cool protein because it fluoresces green and serves as a
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valuable tool for scientists.
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Scientists have found that the protein has a beta barrel structure, a barrel
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shape made up of beta strands that you heard about from Professor Landers.
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Scientists copy the gene encoding GFP and place into the bacteria E. coli.
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So where do we produce this GFP for purification?
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We need to make many copies of the protein to efficiently isolate the
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protein of interest.
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Let's get started.
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I have E. coli cells that are designed to express GFP.
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They grew and divided overnight.
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I also have cells that don't express GFP.
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We'll collect these cells too as a comparison.
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Because it's not ideal to isolate GFP from jellyfish in the lab, we can make
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many copies of GFP by expressing the protein in the bacterium E. coli.
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How do we get bacteria to make protein from a jellyfish?
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Scientists copy the gene encoding GFP and place it in the bacterium E. coli.
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You'll learn more about this process of cloning later in the course, as
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well as how scientists use GFP in research.
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We spin the cells at a high speed in these tubes, so that the cells go to
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the bottom as a pellet.
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How do we know that our bacterial cells really are expressing GFP?
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We can use this really cool property of GFP that it fluoresces.
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I have this UV lamp here.
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And when we shine the UV light on the cells that are expressing GFP, they
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should glow green.
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Let's test our cells.
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I'm removing the liquid from the tube and saving the cell pellets.
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Let's shine the light on the cells that are not supposed to express GFP.
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What do you expect to see?
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Nothing, right?
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No green fluorescence.
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Let's shine the light on the cells that are supposed to express GFP.
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Check it out.
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Green glow.
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So how will we lyse our E. coli cells now to get at that mixture of proteins
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within, including GFP?
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I'm re-suspending the cell pellets in a buffer by pipetting up and down
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repeatedly.
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Now I'm adding an enzyme that breaks down cell walls called lysozyme.
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Next, I'll freeze then thaw the cells quickly.
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This lyses the cells, releasing all the proteins, including
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GFP, from the cells.
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Afterward, I'll centrifuge the samples again to separate insoluble cell
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debris from the soluble proteins.
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So how will we separate GFP from all the other soluble proteins in the
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bacterial cell?
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Now that I have the cell debris separated from the soluble proteins,
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I'll use a small column packed with tiny beads to separate the proteins by
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a specific property.
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In this case, I'm using an affinity column that
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contains hydrophobic beads.
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GFP contains many hydrophobic amino acids.
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In a high-salt buffer, the hydrophobic parts of GFP move to the exterior of
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the protein, and GFP binds to the beads as the
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protein enters the column.
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The other bacterial proteins with fewer hydrophobic amino acids flow
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past the beads and through the column.
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Let's prepare our column for purification.
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I'm adding a buffer with the same salt concentration as our protein solution
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to equilibrate the beads.
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I'm removing the cap at the bottom of the column to allow the
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buffer to flow through.
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With that done, I recap the bottom of the column.
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Now I'm adding my cell extract of soluble proteins and a high salt
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buffer to the top of the column.
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Using this UV lamp, I should be able to detect any GFP.
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Take a peek--
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glowing green!
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So we can see a ring of green fluorescence at the top of the column
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showing that GFP has bound to the top of the column.
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So how will we make sure that all the other bacterial proteins are removed
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from the column?
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I'm adding a wash buffer with a medium salt concentration to our column.
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Any proteins with less hydrophobic exteriors than GFP did not bind as
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tightly to the hydrophobic beads, and will flow out of the
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column during washing.
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The hydrophobic exterior of GFP has a higher affinity for the beads and
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remains bound.
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So now that all bacterial proteins are washed away, how do we remove GFP from
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the beads on the column?
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Remember, the GFP bound to beads in the column because the beads in the
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GFP exterior were currently both hydrophobic.
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I can remove GFP from the beads by adding a buffer with low salt
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concentration.
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So what do you think the low salt concentration solution will do to GFP?
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The low salt concentration will bring the hydrophilic residues of GFP to the
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surface, so the protein will no longer bind to hydrophobic
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beads in the column.
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I'm collecting fractions of what comes off the column in several tubes.
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Let's see if the GFP is moving through the column now with our UV light.
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Yes, I can see the GFP moving through the column.
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Which tube contains the GFP protein?
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I do not know by only looking at the liquid collected off the column.
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For most proteins, we would have to test each fraction with an activity
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assay or another protein detection assay.
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But remember, we're purifying a protein with the
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unique ability to fluoresce.
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Let's check which fraction I collected contains the GFP by using
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the UV light again.
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I can see that most of the green fluorescence is in tube four.
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So GFP eluted in fraction four.
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I've now purified GFP for most of the other bacterial proteins.
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And you've learned something about biochemistry and
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how we purify proteins.
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I hope you had fun.
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