Experiments on the purification of proteins by ion exchange chromatography

Summary

Ion exchange chromatography is the most widely used of the chromatographic means for purifying proteins. It has a high resolution for proteins, is easy to perform, reproducible and low cost. According to the ion exchange principle, proteins can be separated from a large number of buffered solutions, so this method is particularly suitable for the initial purification of protein crude extracts. Speed is often important in the separation of proteins. Examples include the initial separation of proteases and the purification of unstable proteins. Ion exchange chromatography provides many means of accelerating separations. These features make ion exchange chromatography a highly valuable means of protein purification and a highly feasible starting point when designing protein purification protocols. Source: Handbook of Protein Technology

Operation method

ion exchange chromatography

Principle

Proteins that are ion-exchangeable must have a single charge under experimental conditions. Proteins with a single charge in solution can be displaced (meaning exchanged with protein terminal ions) by small ions on the ion exchange resin; immobilized on the resin. Proteins can be eluted from the resin by methods such as increasing the concentration of opposite ions in solution or decreasing the number of charges carried by the protein. Using this method, different proteins can be separated according to their different charge properties. If the isoelectric point of the protein is known, it is easy to formulate a strategy for protein separation. In the case of unknown proteins, a lot of biophysical information about proteins can be obtained from ion exchange pre-tests and guide further purification.

Materials and Instruments

Protein Solutions
Ion exchange chromatography systems

Move

1. Selection of ion exchange resins

Anion exchange resins are used for proteins with a negative net charge; cation exchange resins are used for proteins with a positive net charge. agar-based resins such as DEAD or CM-Sepharose are suitable for many proteins.

1.1 Types of Ion Exchange Resins

In addition to being categorized as anionic or cationic, ion exchange resins are divided into strong and weak ion exchange resins. Strong ion exchange resins are highly ionized over most of the pH range, while weak ion exchange resins are poorly ionized at the working pH. Strong ion exchange resins are used in ion exchange where the pH of the liquid phase is very high or very low. In the past, most chromatographic tests were performed with weak ion exchange resins, but strong ion exchange resins provide better reproducibility.

An important ion exchange resin is hydroxyapatite. It uses calcium phosphate crystals as an ion exchanger and can handle both acidic and basic proteins.

1.2 Chromatography speed

The resin should be shaped so that the flow rate of the liquid phase during chromatography is moderate, allowing sufficient time for the protein to adsorb onto the resin. Too high a chromatographic speed saves time, but also reduces the precision of the test.

1.3 Resin adsorption capacity

Resin adsorption capacity is a standard for evaluating the amount of protein that can be processed by a certain resin, and its unit is mg protein/mL resin. It determines the amount of resin to be used for chromatography. For high-precision chromatography, it is best to use only 10% to 20% of the maximum adsorption capacity of the resin. The adsorption capacity of the same resin is lower for high molecular weight proteins than for low molecular weight proteins because low molecular weight proteins have more binding sites with the resin.

1.4 Resin swelling

Some materials, such as Sephadex, can be compressed by high pressures or swell or shrink when ionic conditions change. Such resins need to be regenerated. Swelling of the resin can complicate handling of the chromatography column. Most newer resin materials have overcome this problem.

1.5 Stability to pH

If the pH value in the experiment is very low or very high, the resin must have a comparable stability. Moreover, the degree of ionization of the weak ion exchange resin will be small, and the strong ion exchange resin should be used.

2. Preparation of resin and filling of chromatographic columns

The preparation of the chromatography column can be divided into three steps: (1) dissolve the dried resin; (2) load the resin into the column; (3) soak the resin with protein-free sample buffer to reach the exchange equilibrium. In order to maintain protein activity, the test can be performed at 4°C. All equipment and reagents should be cooled to 4°C and stored at this temperature.

2.1 Dissolving the Resin

Commercial resins are dried powders and must be solubilized by soaking in sample buffer. 1 g of resin can be solubilized to a volume of 5-50 ml.

2.1.1 Soak the dried resin in about ten times the volume of sample buffer (e.g., 10 g resin + 100 ml sample buffer).

2.1.2 Boil for more than 1 h or soak for several hours to several days to fully moisten the resin. Add more sample buffer during boiling to ensure that the sample buffer is not boiled dry.

2.1.3 Stir the resin several times during the swelling process to ensure that the resin is fully wetted.

2.1.4 Replace the sample buffer several times during the swelling process, so that the composition exchanged into the resin and the sample buffer composition is not very different. Do not use a stirring magnet during the swelling process, as it will break the resin into small pieces.

2.2 Loading the Resin onto the Column

The resin must be equilibrated by soaking it in sample buffer prior to loading the column. Sometimes tens of times the volume of sample buffer is needed to soak the resin, which can save a lot of time. The resin gel used for column loading should be a mixture of one volume of dry resin and one volume of sample buffer. The column height should be 4 to 5 times the column diameter for high-precision chromatography and only 2 times the column diameter for general chromatography. Great care must be taken when loading the column. The column must be loaded in a single pass or the accuracy and reproducibility of the protein separation will be seriously affected. A resin reservoir can provide a sufficient amount of resin for a single column load (see Figure 1).

Figure 1 Resin storage

2.2.1 Wash the resin in a beaker with sample buffer. Stir for 1 min and measure the pH. If the pH is different from the original sample buffer, pour off most of the sample buffer, add new sample buffer, stir again and measure the pH. This step is called partial equilibration and reduces the time it takes for the resin to formally equilibrate in the column;

2.2.2 If chromatography is performed at room temperature, vacuum pump the resin for 1 h to remove small air bubbles, shaking the column while pumping will help to dislodge the bubbles;

2.2.3 Add a small amount of sample buffer to the column;

2.2.4 Open the outlet of the column and export a small amount of sample buffer to drive out the air at the bottom of the column;

2.2.5 Shake the resin well and introduce it into the column with a glass rod without bringing in air bubbles;

2.2.6 opening the outlet of the chromatography column and adding more sample buffer as the resin builds up;

2.2.7 An adapter may be used to load the column. Make certain that no air bubbles enter the column; they can affect the rate of chromatography or even stop the chromatography;

When loading crutches, long chromatography columns can be tilted slightly. It is worth noting. Some resins are better loaded slowly, such resins are dextran gel resins, etc.; while other resins, such as agar gel resins, are best loaded quickly.

2.3 Bringing the resin to exchange equilibrium

After column loading, the resin is finally rinsed with sample buffer to bring its pH and ionic strength up to experimental requirements and to bring the resin to baseline levels. The sample buffer used in this step is typically no more than 10 times the volume of the resin. The pH and conductivity of the sample buffer are compared to the eluent to ensure that the resin has equilibrated.

3. Sample Loading

3.1 Sample Preparation

The sample solution is first clarified by centrifugation or filtration through 0.45 um pore size filter paper. Care should be taken not to lose the target protein during this process. The ionic strength of the sample buffer in which the proteins are solubilized should preferably be less than 50 mmol/L. Gel filtration, dialysis or ultrafiltration can be used for this purpose. Dilution of the protein solution is also an option.

3.2 Sampling

3.2.1 Open the bottom outlet of the column and introduce Sample Buffer into the column until the level reaches the surface of the resin. Close the bottom outlet of the column;

3.2.2 Slowly add the protein solution to the resin surface with a pipette;

3.2.3 Open the outlet of the chromatography column to allow the protein solution to enter the resin, and close the outlet of the column when the surface of the solution coincides with the surface of the resin;

3.2.4 Add a small amount of sampling buffer slowly and dropwise to the resin surface;

3.2.5 Repeat step 3.2.3;

3.2.6 Repeat step 3.2.4 and hang the column;

3.2.7 Protein chromatography and elution can then proceed.

4. Column wash and elution of target proteins

Prior to the elution of any bound proteins, a continuous flow of sample buffer through the ion exchange resin column removes proteins that are not bound to the resin and separates them from proteins adsorbed on the resin. This step typically uses 3 to 10 times the volume of sample buffer as the resin. Testing the effluent for protein concentration or optical density is necessary. Protein elution should begin when trace amounts of protein are present.

As mentioned earlier, protein elution is divided into step elution and gradient elution. In stepwise elution, the increase in ionic strength at each stage need not be consistent, such as when eluting with NaCl solution, which can be eluted in three steps, with NaCl concentrations of 0.1 mol/L, 0.5 mol/L, and 1.0 mol/L in that order (see Fig. 2). The ionic strength of the eluent is gradually increased during the gradient elution, e.g., the NaCl concentration in the eluent is gradually increased from 0.02 mol/L to 1.0 mol/L (see Fig. 3). Gradient elution can facilitate the separation of different proteins more steeply, and stepwise gradient elution can simplify the steps; speed up. However, some proteins cannot be separated when the ionic strength of the stepwise gradient elution varies too much. Gradient elution is necessary for precise protein separation or when two proteins have similar isoelectric points. Stepwise elution may be employed after pre-testing or after the destination protein has been washed out.

4.1 Step elution (Figure 2)

4.1.1 Pass 3 to 10 times the volume of eluent (e.g., 20 mmoo/L Tris-HCl, 0.1 mol/L NaCl solution) through the chromatography crutch.

4.1.2 Make the surface of the eluent flush with the resin bed, so as to ensure that there is no deviation in the change of NaCl concentration when adding different concentrations of eluent.

4.1.3 Change the eluent to the second concentration; repeat steps 4.1.1 and 4.1.2.

Figure 1 Absorbance curve of eluent for stepwise elution.

4.2 Gradient elution

The total amount of column wash buffer for gradient elution should be 5 to 10 times the volume of the resin bed.

4.2.1 Place the lower ionic strength eluent (20 mmol/L Tris-HCl, 0.02 mol/L NaCl) at the end of the gradient closer to the column and the higher ionic strength eluent (20 mmol/L Tris-HCl, 1.0 mol/L NaCl) at the other end. The two containers for the eluent should be the same, and the liquid level in the lyser must be the same. Make sure there are no air bubbles in the gradiometer. Turn on the electromagnetic stirrer in the low concentration eluent container and mount the gradiometer on the chromatography column.

4.2.2 Collect the eluate with a partial collector and check its composition.

Figure 3 Absorbance curve of the eluate for gradient elution

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