Protein Purification: Assays, Specific Activity, Initial Fractionation

互联网2013-09-06

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A successful protein purification procedure can be nothing short of amazing. Whether you are starting off with a recombinant protein which is produced in E. coli , or trying to isolate a protein from some mammalian tissue, you are typically starting with gram quantities of a complex mixture of protein, nucleic acids, polysaccharide, etc. from which you may have to extract milligram (or microgram!) quantities of desired protein at high purity, and hopefully with high yield.

The first step in any purification is the development of a specific assay for the protein of interest

The specific assay can be based upon some unique characteristic of the protein of interest

Enzymatic activity
Immunological activity
Physical characteristics (e.g. molecular mass, spectroscopic properties, etc.)
Biological activity
Ideally, an assay should be
- Specific (you don't want a false positive)
- rapid (you don't want to wait a week for the results)
- sensitive (you don't want to consume all your sample in order to assay it)
- quantitative (you need an accurate way to measure the quantity of your protein at each step in the purification)

Antibodies can be used in a method called Western blotting , which is useful for determining levels of protein expression and for assaying proteins during purification. This method usually involves the following steps:

A protein sample is subjected to polyacrylamide gel electrophoresis.
After this the gel is placed over a sheet of nitrocellulose and the protein in the gel is electrophoretically transfered to the nitrocellulose.
The nitrocellulose is then soaked in gelatin to "block" its ability to non-specifically bind proteins.
The nitrocellulose is then incubated with the specific antibody for the protein of interest.
The nitrocellulose is then incubated with a second antibody which is specific for the first antibody. For example, if the first antibody was raised in rabbits, the second antibody might be termed "goat anti-rabbit immunoglobulin". What this means is that rabbit immunoglobulins were used to elicit an antibody response in goats. The goat antibodies (polyclonal) will include those which recognize the conserved region in the rabbit antibodies. Since the Fc region is conserved, it will bind to any and all rabbit antibodies, including those on the nitrocellulose paper.
The second antibody will typically have a covalently attached enzyme which, when provided with a chromogenic substrate, will cause a color reaction.
Thus the molecular weight and amount of the desired protein can be characterized from a complex mixture (e.g. crude cell extract) of other proteins.

In a variation of the above, the protein sample may be blotted directly on a nitrocellulose paper (called a dot blot ) without first running a gel. This may be desirable if, for example, the antibody is monoclonal and recognizes an epitope which is dependent upon native structure (which would be destroyed upon running an SDS PAGE).

In addition to their varied uses, antibodies can also be used to purify proteins.

If relatively large amounts of an antibody can be obtained, they can be covalently attached to a chromatography resin (e.g. sephadex beads).
If a crude cell extract is run over such a column, only the protein of interest should bind, and everything else will flow through.
The bound protein can then be eluted. This is typically achieved by moderately low pH conditions (using acetic acid). As long as the protein of interest is not irreversibly denatured by such conditions, the method will work quite well.
One potential pitfall involves that of monoclonal antibodies being utilized to purify mutant proteins. The regions of the protein comprising the epitope cannot be modified without destroying the ability of the antibody to bind. Thus, the use of monoclonal antibodies in a purification scheme may preclude its use in purifying certain mutants.

Protein purification can be thought of as a series of fractionation steps designed so that:

The protein of interest is found almost exclusively in one fraction (and with good yield)
A significant amount of the contaminants can be found in a different fraction

During purification you will need to monitor several parameters, including:

Total sample volume
Total sample protein (can be estimated by A₂₈₀ ; 1.4 ~ 1.0 mg/ml)
Units of activity of desired protein (based on specific assay)

This basic information will allow you to keep track of the following information during each step of purification:

% yield for each purification step
Specific activity of the desired protein (units/mg total protein)
Purification enhancement of each step (e.g. "3.5x purification)

In designing a purification scheme you typically have to balance purification with yield .

For example, it may be relatively straightforward to obtain 90% pure material with good yield.
However, it may be difficult to improve that purity an additional few percentile with good yield.
The planned application of the purified protein determines the target purity .
If the protein is to be used to determine amino acid sequence information, maybe 90% is acceptable. However, if the material is to be used in clinical trials, 99.99+% may be the target purity.

Initial steps in purification

It is extremely helpful to have some information not only on the general physical and chemical characteristics of the protein you are trying to purify, but also on the contaminating components.
For example, many E. coli proteins are generally low molecular weight (<50,000 Da) and somewhat acidic in isoelectric point

Usually the initial steps in purification make use of general physical and/or chemical differences between soluble proteins and other cell components.

For example, soluble proteins can be separated from general cellular debris, and intact cells, by centrifugation .
Thus, cells are physically disrupted (via homogenization or a cell press) to allow release of cell contents. This is then followed by centrifugation to separate generally soluble components from those which are insoluble.
It is at this point that data collection begins in order to monitor the purification.

Nucleic acids can sometimes be readily removed from the sample by the addition of large cationic compounds such as polyethylene imine , or streptomycin sulfate .

The nucleic acids bind to these compounds via electrostatic interactions and the complex precipitates and can be removed via centrifugation.
The same general result can be obtained by mixing in ion exchange resins which are anion exchangers (i.e. the resins contain cationic groups) and then filtering or centrifuging to remove. As with either method, it should be confirmed that the desired protein is not bound as well.

Crude fractionations of proteins can be achieved by adding various quantitites of precipitants such as ammonium sulfate , or polyethylene glycol (PEG).

For this type of purification step an initial experiment is performed to monitor the fraction of overall protein, as well as desired protein, remaining in solution (and pellet) as a function of precipitant concentration.

Ammonium Sulfate (% saturated)	0	10	20	30	40	50	60	70	80	90
Sample A₂₈₀	1000	900	600	200	100	75	50	40	25	20
Activity assay(units)	200	200	200	190	170	100	30	5	0	0

In this particular example we are in luck: at around 30% ammonium sulfate we can precipitate about 80% of the total protein concentration in our sample, yet our activity assay for our desired protein indicates that about 95% of our desired protein is still soluble.
At 80% ammonium sulfate all of our desired protein has precipitated. Thus, from these results we would do the following:

Add ammonium sulfate to our sample to a concentration of 30% saturation
Centrifuge and discard the pellet
Add ammonium sulfate to 80% saturation
Centrifuge and keep the pellet. Resuspend the pellet in buffer to solubilize the protein.

We would expect about a 5-fold purification with about 95% yield.