5.6: Introduction to Protein Purification

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 animal or plant tissue, you are typically starting with a complex mixture of proteins, nucleic acids, polysaccharides, lipids, etc. from which you may have to extract milligram (or microgram!) quantities of the desired protein, often with high purity, and hopefully with high yield.

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

Figure 5.6.1: Fractionation

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

The assay can be based upon some unique characteristic of the protein of interest, possibly involving:

• 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 or false negative)
  • 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)

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During purification you will need to keep track of several parameters, including:

1. The total volume of your sample
2. The total amount of protein in your sample

· can be estimated by using E^1%_280nm =14.5

· In other words, measure absorbance at 280nm, divide by 1.4, and you will have the approximate protein concentration in mg/ml

3. The total amount of the protein of interest (the one you are trying to purify). This information will be determined from your quantitative assay

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

1. % yield for each purification step
2. Specific activity of the desired protein ("units of activity" of desired protein/mg total protein)
3. 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 by an additional few percentile and still maintain a 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, 99.999+% may be the target purity.

Initial steps in purification

• Typically (but not always) the protein of interest is contained within the cytoplasm of a cell (sometimes, however, it might be secreted by the cell into the extracellular environment). Thus, cells must be broken open to release the cytoplasm, a process known as cell lysis.
• 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

A typical general outline of a protein purification procedure might look like this:

Figure 5.6.2: Protein purification steps

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 quantities of precipitants such as ammonium sulfate, orpolyethylene 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 (in solution)	1000	900	600	200	100	75	50	40	25	20
Activity assay(units) (in solution)	200	200	200	190	170	100	30	5	0	0

Figure 5.6.3: Protein concentration and precipitant concentration

How can we use this information to utilize ammonium sulfate precipitation as a useful purification step in our overall purification procedure?

Let's begin by calculating the specific activity (a measure of purity) of the starting sample:

Specific activity = total units of desired protein / mg of total protein

To calculate these parameters we need to know the total sample volume (in addition to the information in the above table). In this case, the total sample volume is 1 L. Knowing this, and the absorbance, we can determine the total protein in the sample:

Sample A₂₈₀/1.4 » mg/ml of sample

1000/1.4 = 714 mg/ml

714 mg/ml * 1.0L(1000ml/L) = 714,000 mg total protein

The protein of interest was assayed at 200 units present in the starting sample. Therefore, the specific activity of the starting sample is:

Specific activity = 200 units / 714,000 mg = 2.80 x 10^-4 units/mg

Note

Note: the "units" of activity depend upon the protein of interest. It could be a measure of enzymatic activity - i.e. 1.0 unit of enzymatic activity results in 1mmol of substrate being consumed in 1 min time.

We can now expand upon the above table to include the specific activity of the sample

Ammonium Sulfate (% saturated)	0	10	20	30	40	50	60	70	80	90
Sample A280 (in solution)	1000	900	600	200	100	75	50	40	25	20
Total mg protein (in solution)	714,000	643,000	429,000	143,000	71,400	53,600	35,700	28,600	17,900	14,300
Activity assay(units) (in solution)	200	200	200	190	170	100	30	5	0	0
Specific activity (units/mg)	2.80x10^-4	3.11x10^-4	4.66x10^-4	13.3x10^-4	23.9x10^-4	18.7x10^-4	8.40x10^-4	1.75x10^-4	0	0

We can plot the specific activity of the sample remaining in solution for the different concentrations of added ammonium sulfate:

Figure 5.6.4: Specific activity and precipitant concentration

• The highest specific activity is observed in solution when ammonium sulfate is added to 40% saturation
• What is the increase in purity for the 40% solution sample?

Purification = final specific activity / initial specific activity

Purification = (23.9x10^-4 units/mg) / (2.80x10^-4 units/mg) = 8.5-fold purification

However, purification is not the entire story. We also need to consider yield:

Yield (%) = (final total units / initial total units) x 100

And we can add this information our purification statistics:

Ammonium Sulfate (% saturated)	0	10		20	30	40	50	60	70	80	90
Sample A280 (in solution)	1000	900		600	200	100	75	50	40	25	20
Total mg protein (in solution)	714,000	643,000		429,000	143,000	71,400	53,600	35,700	28,600	17,900	14,300
Activity assay(units) (in solution)	200	200		200	190	170	100	30	5	0	0
Specific activity (units/mg)	2.80x10^-4	3.11x10^-4		4.66x10^-4	13.3x10^-4	23.9x10^-4	18.7x10^-4	8.40x10^-4	1.75x10^-4	0	0
Purification	N/A	1.1	1.7		4.8	8.5	6.7	3.0	0.6	-	-
Yield (%)	100	100	100		95	85	50	15	2.5	0	0

Figure 5.6.5: Percent yield and precipitant concentration

Information on the yield shows that our protein of interest begins to precipitate around 30% ammonium sulfate. Thus, while the addition of ammonium sulfate to 40% provides the greatest purification, it is associated with a 15% loss of our protein of interest. Another option is to added ammonium sulfate to 30%. In this case, we would realize a purification of 4.8-fold, and suffer losses of only 5%.

At each step of a purification scheme we are typically faced with a decision regarding a tradeoff between purity and yield

• If the starting material is cheap, plentiful, or easy to obtain, we may choose our fractionation steps so as to maximize purity at the expense of yield
• If the starting material is expensive, limited, or difficult to get, we may choose to maximize yield at the expense of purity
• Again, these decisions are also determined by the intended purpose of the final material, and therefore, the final purity desired.

Thus, in the above experiment, we would choose to add ammonium sulfate to a concentration between 20-30% if we wanted to maximize yield, or perhaps 40% (or slightly higher) if we wanted to maximize the purification.

Practical matters with ammonium sulfate fractionation steps

We would probably like to remove the ammonium sulfate from our sample (perhaps the next purification procedure cannot tolerate high ionic strength buffers). Notice that from the data in the above table it looks like all our protein of interest is precipitated if ammonium sulfate is added to a concentration of 80%. Thus, this we can use this precipitation property to remove our protein from most of the ammonium sulfate:

1. Add ammonium sulfate to our sample to a concentration of 20-40% saturation (the exact percentage being determined by whether we wish to maximize purity or yield)
2. Centrifuge and discard the pellet
3. Add ammonium sulfate to 80% saturation to the retained solution
4. Centrifuge and keep the pellet. Resuspend the pellet in buffer to solubilize the protein.