All language subtitles for 24-Protein Purification - Beta-galactosidase--en

NATHANIEL: Hi, my name is Nathaniel.

And as a scientist at MIT, I study how ultraviolet light damages the proteins

in the human lens.

And to do so, I purify my human lens proteins from the bacteria E. coli,

using bacteria specifically programmed to express human proteins.

In this demonstration video, I'll show you how biochemists use column

chromatography, fractionation and an enzyme assay to purify the enzyme beta

galactosidase.

In lecture, Professor Lander talked about a number of different enzymes,

like triose phosphate isomerase.

After the end of this video, you'll be able to explain how scientists use

column chromatography and fractionation to purify a number of

different enzymes.

So what does the enzyme beta galactosidase do?

Beta galactosidase hydrolyzes the disaccharide lactose into the

monosaccharides glucose and galactose.

In the bacteria E. coli, beta galactosidase is

encoded by the gene lacZ.

Beta galactosidase breaks down lactose into glucose--

the preferred sugar of E. coli.

So before we get started, let's talk about what we'll be doing today.

How do we purify proteins in the lab?

First, we need to make many copies of the protein.

Then we have to isolate our protein of interest from the other proteins in

the cell that expressed it.

We could make many copies of beta galactosidase by expressing the

protein in the bacterium E. coli.

You'll learn more about how to get the E. coli to express lots of proteins

later in the class.

After we've got many copies of beta galactosidase made, we'll perform

protein purification using column chromatography.

You can see another example of column chromatography in the GFP video.

First, we'll break open the cells to collect all the soluble proteins.

Then we'll use a technique called affinity column chromatography to

separate beta galactosidase from all the soluble bacterial

proteins in the cell.

How does affinity chromatography work?

We take advantage of the unique property in the protein of interest.

In this case, we know that we want to isolate the enzyme beta galactosidase.

Enzymes bind to specific ligands.

The column consists of many tiny beads covalently attached to a ligand that

binds to beta galactosidase.

Because the liquid is clear when we remove beta galactosidase from the

column, we will use a colorimetric test for enzyme activity to determine

where beta galactosidase is present.

So let's get started.

We want our bacterial cells to express a lot of our protein.

To accomplish that, I'm using a string of E. coli cells that constitutively,

or always, expresses beta galactosidase at a high level.

I'm going to collect the E. coli cells that grew in this test tube by

centrifugation.

We spin the cells at a high speed in these tubes so that the cells pellet

at the bottom.

So how do we get the beta galactosidase out of the cells now?

I'm adding a buffer to these cell pellets and resuspending the pellets.

I'm also adding lysozyme, an enzyme that breaks down cell walls.

Combined with the freezing and thawing step, this lyses the E. coli cells,

freeing beta galactosidase.

Afterward, I'll centrifuge the samples again to separate the cell debris from

the soluble proteins.

So now that we have our soluble E. coli proteins and our over-expressed

beta galactosidase, how do we separate out of the cell abstract just the beta

galactosidase that we want from all of the E. coli proteins?

I'm going to add this cell extract to a column to separate the proteins by

affinity chromatography.

In this case, I'm using a small affinity column that contains beads

covalently linked to a ligand that binds beta galactosidase.

This is a ligand that binds the enzyme.

But the enzyme does not act on the ligand.

In research laboratories, we use a number of other columns like this one

to separate proteins based on their size, charge, and other properties.

So let's start the purification process, I'm adding buffer to

equilibrate the column and starting the buffer flowing by removing the cap

at the bottom of the column.

Next, I'm adding my cell extract to the top of the column.

As all the soluble proteins from E. coli cells enter the column, beta

galactosidase will bind to the ligands on the beads.

The other protein should not bind.

So now that the cell extract has fully entered the column, how are we going

to remove all those unwanted proteins from the column?

I'll use this wash buffer to remove unwanted proteins from the column.

So how do we know where beta galactosidase is in the column?

We actually can't tell at this stage.

Unlike when we purified our visibly fluorescent protein like GFP, we can

only test for the presence of most proteins of interest after the

proteins elute from the column.

Now I'll add elution buffer to the top of the column.

The buffer has a much higher pH than the other buffers that we used.

This will push beta galactosidase off the column by disfavoring the

noncovalent interactions between the ligand and the enzyme.

Now I'm going to collect fractions of the liquid coming off the column.

I'm adding elution buffer and continuing to collect fractions.

So now that we've eluted our fractions off of the column, we need some way of

finding out where beta galactosidase ended up, in which fraction.

So we can use beta galactosidase's activity to track where it ended up.

We have a substrate called o-nitrophenyl-be ta-d-galactoside or

ONPG for short.

By tracking which tubes contain a solution that becomes yellow, we can

figure out which fractions contain the most beta galactosidase.

The more yellow that we see indicates the more beta

galactosidase that's present.

I'm adding some of each fraction to a tube containing ONPG solution.

After 10 minutes, I'll add sodium carbonate to make the solution

alkaline, because ONP is yellow in alkaline solutions.

So how will we measure how much yellow product is produced by beta

galactosidase in ONPG?

Well, I'm going to transfer some of our sample to a clear plastic

container called a cuvette, which is transparent to many different

wavelengths of light.

We can use a machine called a spectrophotometer, which measures the

transmittance through a medium at a specific wavelength to figure out

exactly how much yellow product was produced in our sample.

So let's analyze our results.

I got the highest absorbance for the yellow product in fraction four, which

indicates that they've beta galactosidase eluted from the column

in fraction four.

So today you've learned how to purify beta galactosidase from column

chromatography, fractionation, and an enzyme assay.

I hope you had fun.