Hi, guys!
Today, we will talk about the last section of this chapter Protein Purification.
Later, I hope you can get how to purify the proteins you are interested in.
It will be very important for you to do research on proteins in the future.
There some principles of protein purification that you should know.
The basic aim in protein purification is to isolate
one particular protein of interest from other contaminating proteins
so that its structure and/or other properties can be studied.
Because proteins have different distributions in biological materials,
it is important to make the right choice of starting material from
which to purify the protein.
usually be a source that is relatively rich in the protein of interest
and which is readily available.
Once a suitable cellular source of the protein has been identified,
then do the separations.
Separations exploit one or more of the following basic properties of the protein:
its solubility, its size, its charge, its hydrophobicity or its specific binding affinity.
Separations may be chromatographic techniques such as ion exchange,
gel filtration or chromatography,
or electrophoretic techniques such as isoelectric focusing, SDS PAGE.
Of course, it also can base on the basic properties of these proteins
to the combination of fractionation techniques to the Target protein.
However, do you know what’s the Properties of Proteins?
As we have learned, proteins consist of amino acids.
Phe, Tyr and Trp dominate the UV absorption spectra of proteins.
So, proteins have the UV-absorbing properties at 280 nm.
In addition, proteins have isoelectric points (pI).
Acid, base, or protease can catalyze proteins hydrolysis.
And proteins also have colloidal properties.
You know, throughout the protein purification procedure,
steps have to be taken to ensure that the protein of interest
is not inactivated or denatured either by physical or biological factors.
So , next I want to talk about one of the most important properties of proteins, Denature.
we have learned the primary structure of proteins arises from covalent bonds,
the secondary, tertiary,
and quaternary levels of protein structure are maintained by weak, noncovalent forces.
The environment of a living cell is exquisitely suited to maintain these weak forces
and to preserve the structures of its many proteins.
However, a variety of external stresses—for example, heat or chemical treatment
can disrupt these weak forces in a process termed denaturation,
which leading to disruption of the normal 3D structure of a protein,
and loss of protein structure and function.
An example is the denaturation of the protein ovalbumin during the cooking of an egg every day.
About 10% of the mass of an egg white is protein, and 54% of that is ovalbumin.
When a chicken egg is cracked open, the “egg white” is a nearly transparent, viscous fluid.
Cooking turns this fluid to a solid, white mass.
The egg white proteins have unfolded and have precipitated out of solution,
and the unfolded proteins have aggregated into a solid mass.
When denaturation occurs, the following properties of proteins will be changed:
1.Loss of Biological Activity
2.Loss of Solubility
3.Increased Proteolysis
4.More sensitive to protease
5.Increased viscosity
Next, I want to introduce some important experiments
that are used to purify or identify proteins to you.
Let's say you discover a new protein: protein X?
You might ask the following questions:
1.How big is the protein?
2.What is its isoeletric point?
3.What is its structure?
We can get the answers through the following methods
How to get the answers?
We could figure it out through the following methods…..
Here I divided these methods into four categories based on the properties of proteins.
（1） Base on the size of the molecule
① Dialysis
② Gel filtration chromatography
（2） Base on the different solubility
① Isoelectric precipitation
② Ammonium sulfate precipitation
（3）Base on the charge
① Electrophoresis
② Ion exchange chromatography
（4）Base on the ligand specificity --- Affinity chromatography
The first method I will show you is the Dialysis:
this method base on the size of the molecule.
Proteins can be separated from small molecules by dialysis
through a semipermeable membrane such as cellophane(cellulose acetate) .
Pores in the membrane allow molecules up to approximately 10 kDa to pass through,
where as larger molecules are retained inside the dialysis bag.
As mostproteins have molecular masses greater than 10 kDa,
this technique is not suitable for fractionating proteins,
but is often used to remove small molecules such as salts
and ammonium sulfate from a protein solution.
It should be noted that at equilibrium,
the concentration of small molecules inside a dialysis bag will be equal to that outside,
and so several changes of the surrounding solution
are often required to lower the concentration of the small molecule in the protein solution sufficiently.
Now a video for better understanding, real quick, enjoy.
Next method, also base on the size of the molecule，
is Gel filtration chromatography.
In gel filtration chromatography, molecules are separated on the basis of their size and shape.
Small molecules can enter the pores in the beads whereas larger or more elongated molecules cannot.
So, the smaller molecules move more slowly through the column and elute later.
Beads of differing pore sizes are available,
allowing proteins of different sizes to be effectively separated.
And in addition,
Gel filtration chromatography can also be used to estimate the molecular mass of a protein.
There is a linear relationship between the relative elution volume
of a protein and the logarithm of its molecular mass.
Thus a ‘standard’ curve of V e /V o against log 10 molecular mass
can be determined for the column using proteins of known mass.
The elution volume of any sample protein then allows its molecular mass
to be estimated by reference to its position on the standard curve.
Another short video to help you understand.
Ok, another category of protein purification method is Isoelectric precipitation
This technique is based on the solubility
Do you remember the concept of the Isoelectric precipitation, as we known,
when the PH=PI, the amino acid or protein has no net charge, right?
So, proteins will lost the electrostatic repulsion,
and has the lowest solubility at the isoelectric point,
will aggregate and precipitate out at its isoelectric point.
Another common separation step is Ammonium sulfate precipitation, also based on the solubility.
This technique exploits the fact that the solubility of most proteins is lowered at high salt concentrations.
As the salt concentration is increased,
a point is reached where the protein comes out of solution and precipitates.
The concentration of salt required for this salting-out effect varies from protein to protein,
and thus this procedure can be used to fractionate a mixture of proteins.
For example, as shown in this schematic,
protein molecules have colloidal properties and have a layer of hydration membrane on the surface.
In addition, the charge on the surface of the protein produces electrostatic repulsion.
Both of them prevent protein aggregation.
However, when you add the Ammonium sulfate,
it will absorb the Water molecule, destroy the hydrating membrane on the surface of the protein.
Leading to the protein aggregation.
However, many other proteins will also precipitate out at these concentrations of ammonium sulfate.
Therefore this is a relatively crude separation technique,
Salting out is also sometimes used at later stages
in a purification procedure to concentrate a dilute solution
of the protein since the protein precipitates and can then be redissolved in a smaller volume of buffer.
Ok, So How big is protein X? here is a technique you can use,
When placed in an electric field, molecules with a net charge, such as proteins,
will move towards one electrode or the other, a phenomenon known as electrophoresis.
In polyacrylamide gel electrophoresis (PAGE) proteins are applied to a porous polyacrylamide gel
and separated in an electric field on the basis of their net negative charge and their size.
Small/more negatively-charged proteins
migrate further through the gel than larger/less negatively-charged proteins
As shown in this picture, you can see,
In PAGE the electrophoretic separation is carried out in a gel which serves as a molecular sieve.
Small molecules move readily through the pores in the gel,
whereas larger molecules are retarded .
The pore sizes in the gel can be controlled by choosing appropriate concentrations of acrylamide
and the cross-linking reagent, methylene bisacrylamide.
Nowadays, one of the most popular electrophoretic techniques in molecular laboratories is the SDS PAGE.
In SDS-PAGE, full name is SDS-polyacrylamide gel electrophoresis,
the protein sample is treated with a reducing agent to break disulfide bonds
and then with the anionic detergent sodium dodecyl sulfate (SDS)
which denatures the proteins and covers them with an overall negative charge.
The sample is then fractionated by electrophoresis through a polyacrylamide gel.
As all the proteins now have an identical charge to mass ratio,
they are separated based on their mass.
The smallest proteins move farthest.
The steps of this experiment are as follows:
Procedure:
①Incubate protein X with SDS so that it becomes negatively charged
②Load onto gel
③Apply voltage to the gel
④Stain gel with a protein dye (e.g. Coomassie brilliant blue)
SDS-PAGE is a rapid sensitive and widely-used technique
which can be used to determine the degree of purity of a protein sample,
to estimate the molecular mass of an unknown protein
and to deduce the number of polypeptide subunits within a protein.
As shown in this figure,
here is appearance of proteins after electrophoresis on an SDS polyacrylamide .
Lane 1, is proteins (markers) of known molecular mass;
lane 2, is mixture of proteins.
Next video，please
What’s more for electrophoretic techniques. It is Isoelectric focusing
This technique separates proteins on the basis of their relative content of positively and negatively charged groups.
When a protein is at its pI, its net charge is zero and hence it will not move in an electric field.
In isoelectric focusing,
a polyacrylamide gel is used which has large pores
and contains a mixture of polyampholytes
If an electric field is applied to the gel,
the polyampholytes migrate and produce a pH gradient.
Each protein will migrate through the gel until it reaches a position at which the pH is equal to its pI
The steps of this experiment are shown as follows:
①Make a gel with pH gradient
②Load protein
③Find the pH where the protein stops moving
And you can figure out protein X's isoelectric point by this assay.
If you combine SDS-PAGE and isoelectric focusing,
you get 2-dimensional gel electrophoresis (2D gel),
which is very high resolution separations.
Proteins have been separated in the horizontal direction on the basis of their pI,
and in the vertical direction on the basis of their mass.
The overall result is that the proteins are separated both on the basis of their size and their charges.
Thus two proteins that have very similar or identical pIs,
and produce a single band by isoelectric focusing,
if they have different molecular masses will produce two spots by two-dimensional gel electrophoresis.
Similarly, proteins with similar or identical molecular masses,
which would produce a single band by SDS-PAGE,
will also produce two spots if they have different pIs
because of the initial separation by isoelectric focusing.
another technique which can separate proteins, is based on their overall (net) charge.
Let's take a look at schematic illustration of ion exchange chromatography,
if a protein has a net negative charge at pH 7,
it will bind to a column containing positively-charged beads,
whereas a protein with no charge or a net positive charge will not bind (Fig. a).
And the elution diagram (Fig. b) indicates that the negatively-charged proteins bound to such a column
can then be eluted by washing the column
with an increasing gradient of a solution of sodium chloride
at the appropriate pH.
The Cl – ions compete with the protein for the positively-charged groups on the column.
Proteins having a low density of negative charge elute first,
followed by those with a higher density of negative charge.
And you should be wise to that columns containing positively-charged diethylaminoethyl (DEAE) groups
are used for separation of negatively-charged proteins.
This is called anion exchange chromatography.
Columns containing negatively-charged carboxymethyl (CM) groups (such as CM-cellulose or CM-Sephadex),
are used for the separation of positively-charged proteins (cationic proteins).
This is called cation exchange chromatography.
Focus, Another Video is coming.
It’s time to move to the last one, Affinity chromatography
And it mainly exploits the specific, high affinity,
noncovalent binding of a protein to another molecule, the ligand.
As shown in this pic,
First, the ligand is covalently attached to an inert and porous matrix .
And then The protein mixture is passed down a column containing the immobilized ligand.
The protein of interest will bind to the ligand, whereas all other proteins pass straight
through the column
After extensive washing of the column with buffer to remove nonspecifically bound proteins,
the bound protein is released from the immobilized ligand either by adding soluble ligand
which competes with the immobilized ligand for the protein,
or by altering the properties of the buffer (changing the pH or salt concentration).
Because this technique exploits the specific, often unique, binding properties of the protein,
it is often possible to separate the protein from a mixture of hundreds of other proteins in a single
chromatographic step.
Finally, We have conquered Chaper
See you next week.