UNIT 1



UNIT 3

CHROMATOGRAPHY OF PROTEINS

Introduction:

Even when produced by a recombinant host at high expression levels, proteins come in complex mixtures and frequently require multiple purification steps to remove the contaminants. The protein must be separated from hundreds of other proteins with similar properties, as well as all the lipid, nucleic acids, and carbohydrate-containing biomolecules of the cell.

One of the most powerful methods of purification that can do this job is column chromatography. Like all chromatography techniques, there is a stationary phase and a mobile phase in column chromatography. The stationary phase is a fine bead particle that has defined chemical properties that proteins interact with. The mobile phase is a buffer that the protein is soluble in. The separation of proteins on a column relies on their different relative affinities for the stationary, compared to the mobile, phase. Proteins with higher affinity for the stationary phase will be retained on the column while proteins with a greater affinity for the buffer will elute from the column.

There are many types of column chromatography, based on the chemistry of the stationary phase. The general classifications are listed below.

Table 3.1. Types of chromatography commonly used for purifying proteins

|Technique |Based on protein property |

|Gel Filtration (GFP), or Size Exclusion Chromatography (SEC) |Size, shape |

|Ion exchange |Net charge on protein surfaces |

|Hydrophobic interaction chromatography (HIC) |Hydrophobicity of amino side groups on protein surfaces |

|Immobilized metal affinity chromatography (IMAC) |Histidine amino acids on protein surfaces |

|Affinity Chromatography |Biorecognition (ligand specific) |

In the case of SEC, separation on the column is a function of size alone: larger molecules are excluded from the pores of the chromatography beads and elute from the column more quickly than smaller molecules. Here, the resolving power is a function of the length of the column that the proteins are separated on, as well as the pore size of the beads, the flow rate of the elution buffer, and the relative sizes of proteins being separated. Since the permeation of the beads by smaller proteins is a diffusion dependent process, the flow rates should be slow enough to allow for this to happen. In general, the resolution of SEC on small columns is not great, but this step can also be useful for changing the buffer that the protein is dissolved in to the buffer used to elute the proteins from the column. Major drawbacks to SEC is that the volume of the protein sample applied to the column must not exceed 5% of the column volume, and the protein eluted from the column is generally diluted by a factor of 10-fold. This means that a SEC step must usually be followed by a step that will concentrate the protein fraction.

The other forms of chromatography listed in Table 3.1 are a form of “adsorption” chromatography, in that the protein that is applied to the column matrix will be adsorbed to the matrix, allowing separation from proteins that have no affinity to the column. The proteins left on the column can then be eluted by changing the buffering conditions of the mobile phase. A gradual change in buffering conditions can elute proteins one at a time, allowing their separation from each other.

Adsorption chromatography can be used to concentrate proteins, since a large volume of protein can be applied to the column without affecting the amount of protein adsorbed to the column matrix. Careful selection of elution conditions can remove the protein from the column in a much smaller volume. Since adsorption chromatography allow for both the isolation and concentration of a target protein, it is sometimes referred to a “capture” phase of protein purification.

There are different types of materials that chromatography beads are made from. The matrix from which the adsorbent is made has important qualities. If must be readily porous for large proteins, yet sufficiently rigid to sustain hydrostatic pressure of elution buffers. At the same time, the bead material itself must not have a high affinity for proteins: the adsorption of proteins must rely solely on the ligand coating the bead, whether that be an ion exchange group, a hydrophobic group, or an affinity label. The most common chromatographic materials used to make beads are insoluble polysaccharides such as cellulose, dextrans, or agarose. Sephadex is a bead-formed gel prepared by cross-linking dextran with epichlorohydrin. The dextran hydroxyl group renders the gel extremely hydrophilic, and the degree of cross-linking determines the pore size of the gel. The lower the cross-linking, the more porous the bead is and the larger the proteins that have access to its surfaces, both interior and exterior. Unfortunately, the more porous bead have less mechanical strength and are compressed by high hydrostatic pressures found in large columns or when elution buffers are pumped too quickly. Cross-lined agarose-type beads, such as Sepharose, have greater mechanical stability and are preferred for larger columns of for faster elution rates. Both types of beads are biodegradable, so must be stored with a biocide such as sodium azide or high concentrations of alcohol. Although cold storage is best, the beads are damaged by freezing.

There is no single best way to purify all proteins. Finding an optimal protein purification strategy requires trial and error, because the best method for purification depends on the properties of protein being isolated as well as the proteins and other contaminants that the protein must be isolated from. Different chromatography methods are tested and compared for their suitability in purifying each new protein. In choosing the best types of chromatography to use, there are at least five factors that must be evaluated:

□ resolution of the method

□ capacity of the method

□ speed of the method

□ % recovery of protein

□ cost

In this lab exercise, we will compare HIC with an affinity chromatography technique, immobilized metal affinity chromatography (IMAC) in the purification of the GFP that you isolated in Lab Exercise 2.

Hydrophobic interaction chromatography (HIC)

Separation by HIC is based on the reversible interaction between a protein and the hydrophobic surface of a chromatographic medium. This interaction is enhanced at high ionic strength buffer solution (e.g. 1.5 M ammonium sulphate). Proteins bind to the column as they are loaded, and proteins with low affinity and other contaminants are washed from the column with high ionic strength buffer. Conditions are then altered so that the bound substances are eluted differentially, usually by decreasing the salt concentration of the elution buffer. Changes are made stepwise or with a continuously decreasing salt gradient. Target proteins are concentrated during binding and collected in a purified, concentrated form. Some proteins with extremely high affinity to the HIC matrix must be eluted with buffers of reduced polarity (e.g. an ethylene glycol gradient up to 50%). Some will elute by adding chaotropic species (urea, guanidine hydrochloride) or detergents. Sometimes a change of pH or temperature can be used to affect the elution of proteins, as well. In general, hydrophobic interactions increase in strength with increasing temperature; there can be a 20-30% reduction in binding when the temperature is changed from 20 to 4oC. Generally, the strength of the interaction between proteins and hydrophobic ligands decreases with increasing pH. This is presumably because of an increase in the hydrophilicity of proteins due to the titration of charged groups. The effect of pH is different for different proteins, and thus elution profiles may be improved by changing the elution pH. However, the effect of pH on elution from HIC is not that great, and it is best to work with the pH range that the protein is stable at.

The most popular HIC resins are cross-linked agarose gels to which hydrophobic ligands have been covalently attached. The choice of ligand will determine the degree of hydrophobicity of the HIC resin. The most popular ligands include, in the order of increasing hydrophobicity:

methyl- < phenyl- < octyl-.

If a protein binds too tightly, requiring a harsh elution buffer, a less hydrophobic matrix might work better.

A variety of salts may be used for loading and elution. In general the effects of various salts on HIC mimic their effects on salting out proteins. Those salts which are most effect in salting out are most effective at binding proteins to hydrophobic matrices. Thus, ammonium sulfate is generally a good salt for binding most proteins to an HIC column.

Immobilized Metal Affinity Chromatography (IMAC)

IMAC stationary phases are designed to chelate metal ions that have selectivity for specific groups on protein surfaces. A strong chelating group is added to the chromatographic matrix, such that when the metal ions are introduced, they are complexed leaving part of the coordination sphere of the metal ion free. Ligands will therefore coordinate with these metals, including residues in proteins. Histidine, and to a lesser extent cysteine and tryptophan, residues on the protein’s surface are mainly responsible for the interaction with IMAC residues. Most proteins have these amino acid residues, and in some cases a single histidine in a small protein is sufficient to cause an interaction strong enough to bind the protein to an IMAC column.

The nature of the metal and the way it is coordinated on the column also influences the strength of interactions, so that a whole range of selectivity is introduced. Most work has used divalent metals in the first transition series through zinc. Although much of the earlier work concentrated on the use of Cu+2 and Zn+2 complexes, Co+2, Ni2+, and even Fe3+ complexes are commonly used today.

Operation of an IMAC column involves first “charging up” the chelating adsorbent with metal; a small pulse of quite concentrated (50 mM) metal salt is passed through, followed by water. The column acquires a color as themetal ions attach (except for zinc). A prewash with intended elution buffers is usually carried out. Sample buffers contain salt (up to 1 M) to minimize nonspecific ion-exchange effects, and adsorption of proteins is maximal at higher pH. Elution is normally either by lowering of pH to protonate the donor histidine groups on the adsorbed protein, or by use of a stronger complexing agent such as imidazole. Because some eluting buffers also cause some leakage of metal ions, it can be a good idea to have some adsorbent that has not been charged with the metal on the bottom of the column to trap this leakage.

| |

|Safety Considerations: |

|Wear closed toed shoes whenever you are in lab. |

|Wear gloves while handling the acid to adjust the pH. |

|Wear gloves when handling hazardous chemicals and dispose in hazardous waste bin. |

MEDIA PREP FORM Control #

Name of Solution/Media:

Amount prepared: Date:

Preparers(s):

|Component |Brand/Item # |Storage conditions/ |FW or initial |Amount used |Final concentration |

| | |date received |concentration | | |

| | | | | | |

| | | | | | |

| | | | | | |

| | | | | | |

| | | | | | |

| | | | | | |

| | | | | | |

| | | | | | |

| | | | | | |

| | | | | | |

| | | | | | |

| | | | | | |

|Balance used |Calibration status |

| | |

|pH meter used |Calibration status |

| | |

|Initial pH |Final pH |Adjusted pH with |

| | | |

|Prep temperature |Sterilization procedure |Storage conditions |

| | | |

Calculations/Comments:

MEDIA PREP FORM Control #

Name of Solution/Media:

Amount prepared: Date:

Preparers(s) :

|Component |Brand/Item # |Storage conditions/ |FW or initial |Amount used |Final concentration |

| | |date received |concentration | | |

| | | | | | |

| | | | | | |

| | | | | | |

| | | | | | |

| | | | | | |

| | | | | | |

| | | | | | |

| | | | | | |

| | | | | | |

| | | | | | |

| | | | | | |

| | | | | | |

|Balance used |Calibration status |

| | |

|pH meter used |Calibration status |

| | |

|Initial pH |Final pH |Adjusted pH with |

| | | |

|Prep temperature |Sterilization procedure |Storage conditions |

| | | |

Calculations/Comments:

Protocol:

FIRST DAY

On the first day, you will need to set up your solutions and columns for protein purification. There are 3 different column purifications that will be compared; each group will perform one purification step, and comparisons will be made between the different groups in the classroom. All purifications must be included in the lab report in order to make comparisons, so information must be exchanged between groups to be included in individuals’ reports. Students may collaborate in the preparation of the following solutions for use by the entire class. Part of this collaboration is the sharing of the Media Prep Form describing the preparation of each solution in for individual lab reports. Students using a solution should record its control number in their lab report.

1. The following reagents will be used for purification of GFP by chromatography. Groups may share responsibility for making the different reagents, but everyone must have a copy of the prep form for each solution that is used during the experiment. Make sure you double check your calculations with your partners. When finished, dispense the solution into bottles and label each with the reagent, your initials and the date. Make out a Media Prep form. Make up the stock solutions first, and make the working solutions from the stocks when necessary.

|Solution |Final concentration |Final volume |Comments |

|Ammonium sulfate stock solution |4.0 M |100 mL |Store at room temperature |

| | | | |

|Polyethylene glycol (PEG) |5% (w/v) |20 mL |Store at room temperature. |

|Sodium phosphate in purified H2O, |1.0 M |100 mL |Store refrigerated. |

|pH 7.0 | | |Prepared in Lab 2A |

|1M NaCl stock solution (50 mL) |1.0 M |50 mL |Store at room temperature. |

|IMAC equilibration buffer |20 mM Na phosphate |100 mL |From stocks. Store refrigerated. |

| |0.5 M NaCl | | |

| |pH 7.0 | | |

|IMAC elution buffer #1 |0.1 M Na acetate |20 mL |Start with 0.1M acetic acid, adjusting pH with NaOH. Add|

| |0.5 M NaCl | |NaCl from stock. Store refrigerated. |

| |pH 4.0 | | |

|IMAC elution buffer #2 |20 mM Na phosphate |20 mL |Dilute phosphate buffer from stock. Add the imidazole |

| |0.4 M imidazole | |and adjust the pH before adjusting the final volume. |

| |pH 7.0 | |Store refrigerated. |

|HIC equilibration buffer |20 mM Na phosphate |100 mL |From stocks. Store refrigerated. |

| |2 M ammonium sulfate | | |

| |pH 7.0 | | |

|HIC elution buffer #1 |20 mM Na phosphate, 1 M ammonium |20 mL |From stocks. Store refrigerated. |

| |sulfate | | |

| |pH 7.0 | | |

|HIC elution buffer #1 |20 mM Na phosphate, 1 % PEG |20 mL |From stocks. Store refrigerated. |

| |pH 7.0 | | |

2. Set up your columns. These resins are high-capacity, and you should not need more than 2-3 mL of column bed volume. Select your column size accordingly. Fix your column to a ring stand so that the column is vertical. Plumb your column with the narrowest tubing that you can fit to it. If possible, find a stop cock for the bottom fitting so that you can turn off your column when necessary. If you do not have a stock cock for your column, you will need clamps to stop the flow of buffer through your column. You will need to place your buffers above your column to feed through the column by gravity flow.

SECOND DAY

You will compare two types of chromatography with small testing columns, one with HIC phenyl Sepharose resin (phenyl agarose may be substituted) and two with a nickel IMAC resins (with two different types of elution strategies). After packing columns with resins in the appropriate equilibration buffer, you will apply your cell extract to the column to adsorb the proteins. After nonadsorbed proteins and other contaminants are washed from the column with equilibration buffer, proteins will be eluted by gradually changing the elution buffer. You will be able to immediately tell where the GFP is at any point by simply looking for fluorescence under a black light. Fractions containing GFP will be saved for later analysis by gel electrophoresis.

Part I. HIC: Phenyl Sepharose Chromatography

1. Phenyl Sepharose is stored in a sodium azide solution to prevent microbial growth, and the sodium azide must be removed prior to its use. Dispense the appropriate amount of resin by repeatedly inverting the phenyl Sepharose container to resuspend the beads and immediately pipetting or pouring off as much as you will need for this lab (about 5-10 mL per column). Transfer into a 15 mL conical centrifuge tube. Measurements do not have to be exact.

2. Allow the suspension to resettle. This should take 5 minutes or less. You may centrifuge the mixture for a minute in a clinical centrifuge to settle the beads faster.

3. Remove the sodium azide used to preserve the phenyl Sepharose by pipetting it off the top. Be careful to draw up only liquid.

4. Add 5 mL of HIC equilibration buffer (2M ammonium sulfate in 20 mM Na phosphate pH 7.0) and mix well by gentle inversion. This removes most, but not all, of the sodium azide. The rest will be removed in subsequent steps.

5. Allow the beads to settle or centrifuge to settle the beads.

6. Remove the buffer from the top and add back fresh equilibration buffer. This should remove all of the sodium azide that was used as a preservative for the resin.

7. Prepare a column for assembly by clamping a BioRad Poly-Prep column to a ring stand. (If devising your column from a pasteur pipette, use the tip of a straightened large paper clip to push a small plug of glass wool into the tip of a pasteur pipette or dropper to form a “frit” for holding resin in the column. Do not push the glass wool too tight or it will slow the flow of buffer through the column. Also, if you have no ring stand and clamps, you can hold the column in a vertical position by using a styrofoam cup with the sides cut out as a stand. If you have a ring stand but not clamps, use twist ties to affix the column to a ring stand.)

8. Place a 50 mL beaker or plastic cup under the column to collect waste liquids. Keep collecting liquids in this container until the instructions call for collecting column fractions.

9. Use a transfer pipette or pasteur pipette to add approximately 1 mL of equilibration buffer to the column to soak the frit (or glass wool plug) first in order to remove any air bubbles. A standard black dropper bulb draws up about 1 mL. (If the liquid doesn’t start to drop out of the pipette you have pushed the glass wool plug in too tightly. Start again.)

10. As the equilibration buffer is flowing through the frit and before the column goes dry, load your phenyl Sepharose resin. Gently invert the phenyl Sepharose slurry that you have prepared in order to resuspend the beads. With the tip of the pasteur pipette or dropper touching the side of the column, slowly add about 8 mL of slurry. Avoid introducing any air bubbles into the column. The excess liquid will drain through the column and into the waste container beaker below.

11. Let the equilibration buffer flow through the column to equilibrate the column conditions and to pack the column with beads. DO NOT ALLOW THE COLUMN TO GO DRY AT ANY TIME! Make sure the bed height of the column is about 4-5 cm and the top of the column bed is level. The exact height is not important, but make sure that your beads fill the lower, more constricted end of the column. You need enough resin in the column to bind all of the protein molecules you will be loading. If there are more protein molecules than resin beads available to bind them the extra proteins will pass right through the column without being separated. If the column is too short after packing, add more slurry.

12. The general rule in equilibrating columns is to add 2 column volumes of equilibration buffer and let it wash through. Gently load 10 mL of equilibration buffer onto the column without introducing air to the buffer and without disturbing the column bed. You can do this by placing the tip of the pipette or dropper on the wall of the column near to top of the column bed and allowing the buffer to flow gently down the inside of the column. If there is not enough room to add all of the buffer at once, keep adding more as it drains through the column. Allow the buffer to drain through the matrix into the beaker or cup.

13. While your column is equilibrating, prepare your cell extract for loading. It must be thawed and adjusted to 2M in ammonium sulfate. Transfer 0.5 mL of the cell extract to a fresh microcentrifuge tube and add an equal volume of 4 M ammonium sulfate. Any precipitated matter that may have formed from the freeze-thaw procedure or the addition of ammonium sulfate must be removed by centrifugation (full speed 10 minutes in refrigerated micrcentrifuge). If there is no visible precipitation, then there is no need to centrifuge the samples. Carefully transfer the sample supernatant or filtrate to a fresh microcentrifuge tube, using a pasteur pipet. Keep your cell extract cold by placing it on crushed ice.

14. When the last of the equilibration buffer has entered the column, slowly load 1.0 mL of your cell extract onto the top of column. Be careful not to disturb the chromatography bed or to introduce air bubbles.

15. When the cell extract has entered the column bed, carefully add 5 mL of equilibration buffer to the top and collect the column effluent into a test tube marked “wash”, You should see the GFP getting “trapped” in the first layers of the column by observing the column under a black light. This is because the high salt concentration in the equilibration buffer increases the hydrophobic interactions between the proteins in the sample with the phenyl groups of the resin. They are attracted to the hydrophobic beads in the column resin and bind to them.

16. When the wash buffer has eluted from the column, add the 5 mL of 1.0 M ammonium sulfate elution buffer (HIC Elution Buffer #1) that you have prepared to the column. Be careful not to disturb the chromatography resin and be careful not to allow the column to go dry. At this point, you do not know where the GFP will elute, so you need to start collecting fractions for later analysis. Collect 1.5 mL fractions into microcentrifuge tubes. Label your collection fractions by number and store them on crushed ice.

17. When the HIC Elution Buffer #1 has eluted from the column, repeat the elution process using the 5 mL of the HIC Elution Buffers #2. Continue to collect 1.5 mL column fractions in microcentrifuge tubes.

18. View your collected column fraction under UV light to locate the fraction(s) containing GFP. Label the fraction with its contents, the date, and your group, and freeze for later analysis.

Part II. IMAC: Nickel Immobilized Affinity Chromatography with Imidazole Gradient Elution

1. The nickel immobilized chromatography resin is stored in an alcohol solution to prevent microbial growth, and the alcohol must be removed prior to its use. Dispense the appropriate amount of resin by repeatedly inverting the container to resuspend the beads and immediately pipetting or pouring off as much as you will need for this lab (about 10 mL per column). Transfer into a 15 mL conical centrifuge tube. Measurements do not have to be exact.

2. Allow the suspension to resettle. This should take 5 minutes or less. You may centrifuge the mixture for a minute in a clinical centrifuge to settle the beads faster.

3. Remove the ethanol used to preserve the chromatography beads by pipetting it off the top. Be careful to draw up only liquid.

4. Add 5 mL of IMAC equilibration buffer (20 mM Na phosphate, 0.5 M NaCl pH 7.0) and mix well by gentle inversion. This removes most, but not all, of the ethanol. The rest will be removed in subsequent steps.

5. Allow the beads to settle or centrifuge to settle the beads.

6. Remove the buffer from the top and add back fresh equilibration buffer. This should remove all of the ethanol that was used as a preservative for the resin.

7. Prepare a column for assembly by clamping a BioRad Poly-Prep column to a ring stand. (If devising your column from a pasteur pipette, use the tip of a straightened large paper clip to push a small plug of glass wool into the tip of a pasteur pipette or dropper to form a “frit” for holding resin in the column. Do not push the glass wool too tight or it will slow the flow of buffer through the column. Also, if you have no ring stand and clamps, you can hold the column in a vertical position by using a styrofoam cup with the sides cut out as a stand. If you have a ring stand but not clamps, use twist ties to affix the column to a ring stand.)

8. Place a 50 mL beaker or plastic cup under the column to collect waste liquids. Keep collecting liquids in this container until the instructions call for collecting column fractions.

9. Use a transfer pipette or pasteur pipette to add approximately 1 mL of equilibration buffer to the column to soak the frit (or glass wool plug) first in order to remove any air bubbles. A standard black dropper bulb draws up about 1 mL. (If the liquid doesn’t start to drop out of the pipette you have pushed the glass wool plug in too tightly. Start again.)

10. As the equilibration buffer is flowing through the frit and before the column goes dry, load your nickel immobilized chromatography resin. Gently invert the slurry that you have prepared in order to resuspend the beads. With the tip of the pasteur pipette or dropper touching the side of the column, slowly add about 8 mL of slurry. Avoid introducing any air bubbles into the column. The excess liquid will drain through the column and into the waste container beaker below.

11. Let the equilibration buffer flow through the column to equilibrate the column conditions and to pack the column with beads. DO NOT ALLOW THE COLUMN TO GO DRY AT ANY TIME! Make sure the bed height of the column is about 4-5 cm and the top of the column bed is level. The exact height is not important, but make sure that your beads fill the lower, more constricted end of the column. You need enough resin in the column to bind all of the protein molecules you will be loading. If there are more protein molecules than resin beads available to bind them the extra proteins will pass right through the column without being separated. If the column is too short after packing, add more slurry.

12. The general rule in equilibrating columns is to add 2 column volumes of equilibration buffer and let it wash through. Gently load 10 mL of equilibration buffer onto the column without introducing air to the buffer and without disturbing the column bed. You can do this by placing the tip of the pipette or dropper on the wall of the column near to top of the column bed and allowing the buffer to flow gently down the inside of the column. If there is not enough room to add all of the buffer at once, keep adding more as it drains through the column. Allow the buffer to drain through the matrix into the beaker or cup.

13. While your column is equilibrating, prepare your cell extract for loading. It must be thawed and any precipitated matter that may have formed from the freeze-thaw treatment must be removed by centrifugation (full speed 10 minutes in refrigerated micrcentrifuge). If there is no visible precipitation, then there is no need to centrifuge the samples. Carefully transfer the sample supernatant or filtrate to a fresh microcentrifuge tube, using a pasteur pipet. Keep your cell extract cold by placing it on crushed ice.

14. When the last of the equilibration buffer has entered the column, slowly load 0.5 mL of your cell extract onto the top of column. Be careful not to disturb the chromatography bed or to introduce air bubbles.

15. When the cell extract has entered the column bed, carefully add 5 mL of equilibration buffer to the top slowly so that you do not disturb the column bed. Collect the column effluent into a test tube marked “wash”. You should see the GFP getting “trapped” in the first layers of the column by observing the column under a black light. Watch the column so that you do no allow the column to go dry.

16. While you are washing impurities from the column with equilibration buffer, prepare a couple of solutions (5 mL each) of increasing imidazole concentration for eluting adsorbed proteins from the column by a step gradient. You can make the following solutions by diluting IMAC equilibration buffer with the IMAC elution buffer #2 (0.4 M imidazole in 20 mM Na phosphate pH 7.0) to make to following concentrations of imidazole:

□ 0.15 M imidazole

□ 0.3 M imidazole

17. When the wash buffer has eluted from the column, carefully add the 5 mL of the 0.15 M imidazole elution buffer that you have prepared to the top of the column. Be careful not to disturb the chromatography resin and be careful not to allow the column to go dry. At this point, you do not know where the GFP will elute, so you need to start collecting fractions for later analysis. Collect 1.5 mL fractions into microcentrifuge tubes. Label your collection fractions by number and store them on crushed ice.

18. When the 0.15 M imidazole elution buffer has eluted from the column, repeat the elution process using the 5 mL of 0.3 M imidazole elution buffer. Be careful not to disturb the column bed and to not allow the column to go dry. Continue to collect 1.5 mL column fractions in microcentrifuge tubes.

19. View your collected column fraction under UV light to locate the fraction(s) containing GFP. Label the fraction with its contents, the date, and your group, and freeze for later analysis.

Part III. IMAC: Nickel Immobilized Affinity Chromatography with pH Gradient Elution

1. The nickel immobilized chromatography resin is stored in an alcohol solution to prevent microbial growth, and the alcohol must be removed prior to its use. Dispense the appropriate amount of resin by repeatedly inverting the container to resuspend the beads and immediately pipetting or pouring off as much as you will need for this lab (about 10 mL per column). Transfer into a 15 mL conical centrifuge tube. Measurements do not have to be exact.

2. Allow the suspension to resettle. This should take 5 minutes or less. You may centrifuge the mixture for a minute in a clinical centrifuge to settle the beads faster.

3. Remove the ethanol used to preserve the chromatography beads by pipetting it off the top. Be careful to draw up only liquid.

4. Add 5 mL of IMAC equilibration buffer (20 mM Na phosphate, 0.5 M NaCl pH 7.0) and mix well by gentle inversion. This removes most, but not all, of the ethanol. The rest will be removed in subsequent steps.

5. Allow the beads to settle or centrifuge to settle the beads.

6. Remove the buffer from the top and add back fresh equilibration buffer. This should remove all of the ethanol that was used as a preservative for the resin.

7. Prepare a column for assembly by clamping a BioRad Poly-Prep column to a ring stand. (If devising your column from a pasteur pipette, use the tip of a straightened large paper clip to push a small plug of glass wool into the tip of a pasteur pipette or dropper to form a “frit” for holding resin in the column. Do not push the glass wool too tight or it will slow the flow of buffer through the column. Also, if you have no ring stand and clamps, you can hold the column in a vertical position by using a styrofoam cup with the sides cut out as a stand. If you have a ring stand but not clamps, use twist ties to affix the column to a ring stand.)

8. Place a 50 mL beaker or plastic cup under the column to collect waste liquids. Keep collecting liquids in this container until the instructions call for collecting column fractions.

9. Use a transfer pipette or pasteur pipette to add approximately 1 mL of equilibration buffer to the column to soak the frit (or glass wool plug) first in order to remove any air bubbles. A standard black dropper bulb draws up about 1 mL. (If the liquid doesn’t start to drop out of the pipette you have pushed the glass wool plug in too tightly. Start again.)

10. As the equilibration buffer is flowing through the frit and before the column goes dry, load your nickel immobilized chromatography resin. Gently invert the slurry that you have prepared in order to resuspend the beads. With the tip of the pasteur pipette or dropper touching the side of the column, slowly add about 8 mL of slurry. Avoid introducing any air bubbles into the column. The excess liquid will drain through the column and into the waste container beaker below.

11. Let the equilibration buffer flow through the column to equilibrate the column conditions and to pack the column with beads. DO NOT ALLOW THE COLUMN TO GO DRY AT ANY TIME! Make sure the bed height of the column is about 4-5 cm and the top of the column bed is level. The exact height is not important, but make sure that your beads fill the lower, more constricted end of the column. You need enough resin in the column to bind all of the protein molecules you will be loading. If there are more protein molecules than resin beads available to bind them the extra proteins will pass right through the column without being separated. If the column is too short after packing, add more slurry.

12. The general rule in equilibating columns is to add 2 column volumes of equilibration buffer and let it wash through. Gently load 10 mL of equilibration buffer onto the column without introducing air to the buffer and without disturbing the column bed. You can do this by placing the tip of the pipette or dropper on the wall of the column near to top of the column bed and allowing the buffer to flow gently down the inside of the column. If there is not enough room to add all of the buffer at once, keep adding more as it drains through the column. Allow the buffer to drain through the matrix into the beaker or cup.

13. While your column is equilibrating, prepare your cell extract for loading. It must be thawed and any precipitated matter that may have formed from the freeze-thaw treatment must be removed by centrifugation (full speed 10 minutes in refrigerated micrcentrifuge). If there is no visible precipitation, then there is no need to centrifuge the samples. Carefully transfer the sample supernatant or filtrate to a fresh microcentrifuge tube, using a pasteur pipet. Keep your cell extract cold by placing it on crushed ice.

14. When the last of the equilibration buffer has entered the column, slowly load 0.5 mL of your cell extract onto the top of column. Be careful not to disturb the chromatography bed or to introduce air bubbles.

15. When the cell extract has entered the column bed, carefully add 5 mL of equilibration buffer to the top, slowly so that you do not disturb the column bed. Collect the column effluent into a test tube marked “wash”. You should see the GFP getting “trapped” in the first layers of the column by observing the column under a black light. Watch the column to make sure that it does not go dry.

16. While you are washing impurities from the column with equilibration buffer, prepare a couple of solutions (5 mL each) of decreasing pH for eluting adsorbed proteins from the column by a step gradient. You can make the following solutions by diluting IMAC equilibration buffer with the IMAC elution buffer #1 (0.1 M Na acetate, 0.5 M NaCl pH 4.0) in the following ratios:

|Elution gradient step |IMAC equilibration buffer |IMAC elution buffer #1 |

|First |2.5 mL |2.5 mL |

|Second |1 mL |4 mL |

17. When the wash buffer has eluted from the column, add the 5 mL of the first elution gradient step buffer that you have prepared to the column. Be careful not to disturb the chromatography resin and be careful not to allow the column to go dry. At this point, you do not know where the GFP will elute, so you need to start collecting fractions for later analysis. Collect 1.5 mL fractions into microcentrifuge tubes. Label your collection fractions by number and store them on crushed ice.

18. When the first elution gradient buffer has eluted from the column, repeat the elution process using the 5 mL of second elution gradient buffer. Continue to collect 1.5 mL column fractions in microcentrifuge tubes.

19. When the second elution gradient buffer has eluted from the column, repeat the elution process using 5 mL of IMAC elution buffer #1. Continue to collect 1.5 mL column fractions in microcentrifuge tubes.

20. View your collected column fraction under UV light to locate the fraction(s) containing GFP. Label the fraction with its contents, the date, and your group, and freeze for later analysis.

ALTERNATIVE ANALYSIS (if time):

A more quantitative approach to analyzing which chromatography fractions contain GFP would be to determine fluorescence in a fluorimeter. This can be used to determine the following results:

a. How efficient your elution conditions were at releasing GFP from the column in a narrow fraction, producing a small volume of highly concentrated GFP.

b. The quantitative yield of GFP from the column. This can be determined by the fluorescence multiplied by the total volume of GFP collected from the column. The percent yield can be done by comparing the fluorescence of the sample loaded multiplied by the volume of sample loaded onto the column.

CLEAN UP

Chromatographic matrices are very expensive and with proper care, can be reused for an indefinite period of time. In order to properly care for the column matrix, you should “clean up” any tightly adsorbed proteins from the column with an extreme-pH buffer, when possible. Many matrices cannot withstand such a harsh treatment, so make sure that you do not use this approach unless the product literature condones it. It is very important to thoroughly remove this highly acidic or basic buffer from the column before storage.

Since most chromatographic matrices used for protein purifications are made from highly biodegradable materials such as dextrans, it is very important that you store these materials in a bacteriostatic buffer. Microbial growth can be effectively inhibited with a high concentration of ethanol or a dilute solution of sodium azide. Be sure to check the product literature for your chromatographic matrix to decide the best way to preserve your material.

1. Phenyl Sepharose columns: run 5 mL of 10mM NaOH through the column, followed by 20 mL of deionized water. Allow the column to go dry and dispense the beads into a plastic bottle by inverting it and rinsing it with a wash bottle or syringe of deionized water. Adjust the volume with ethanol to make it 20% ethanol. Label the bottle as used, washed phenyl Sepharose. Include your names and date on the label, and place in the refrigerator for long-term storage.

2. IMAC columns: run 20 mL of deionized water through the column. Allow the column to go dry and dispense the beads into a plastic bottle by inverting it and rinsing it with a wash bottle or syringe of deionized water. Adjust the volume with ethanol to make it 20% ethanol. Label the bottle as used, washed nickel-IMAC. Include your names and date on the label, and place in the refrigerator for long-term storage.

Questions for Unit 3

Read Chapter 27 “Introduction to Bioseparations” by Seidman & Moore in Basic Laboratory Mehods for Biotechnology, section II. Chormatography on pp 584-588. Answer the following questions.

a. Chromatography is an expensive purification step. Explain why it is commonly used in the purification of proteins.

b. What are the mobile and stationary phases of the chromatography columns that were used in this lab exercise?

c. What is the basis of protein separations in the chromatography columns that were used in this lab exercise?

d. What is HPLC, and what are some advantages of HPLC chromatography, compared to the columns that you ran in this exercise?

e. What gives rise to the higher resolution of purifications in HPLC?

f. Why do HPLC columns run at such high pressures?

g. What are some reasons that a protein purification strategy should be designed to have the fewest possible number of purification steps?

h. What measures should be taken to stabilize proteins during purification procedures?

i. What are some special considerations that must be considered when scaling up a protein purification procedure into a production facility?

Compare the results from the three column purifications that were investigated by filling out the following table.

|Column purification |Fraction(s) where GFP eluted |Volume of GFP fraction |Relative fluorescence of GFP |

| | | |fraction |

|HIC-phenyl Sepharose | | | |

|IMAC-imidazole elution | | | |

| | | | |

|IMAC-pH gradient elution | | | |

| | | | |

1. Which procedure had the best results? Explain your reasoning, discussing the relative GFP concentrations and the relative volumes that the GFP eluted in. Remember that the goals of chromatography include a high overall yield as well as a concentration of the molecule that you wish to purify.

2. What might you do to improve the purification of GFP by HIC and IMAC, based on the class results?

3. Go the National Center for Biological Information (NCBI) at ncbi.nlm. and search for the amino acid sequence of the green fluorescent protein.

a. How many amino acids are there in the GFP?

b. What amino acids, in the GFP, are hydrophobic?

c. What percentage of the GFP amino acids is hydrophobic?

References

Chalfie, Martin and Steven Kain, Green Fluorescent Protein, Properties, Applications and Protocols Wiley-Liss, 1998.

Doonan, S (ed.) Protein Purification Protocols in Methods in Molecular Biology (Vol. 59) Humana Press (1996)

Price, N,C, (Ed.) Proteins Labfax. Academic Press. (1996)

Scopes, R.K. Protein Purification: Principles and Practice (2nd ed) Springer-Verlag (1987)

Sommer, C.A., F.H. Silva, and M.T.M. Novo. Teaching Molecular Biology to Undergraduate Biology Students. Bichem & Mol Biol Ed. 32(1):7-10 (2004)

Walmsley, R.M., N. Billinton, and W.-D. Heyer. Yeast functional analysis report: green fluorescent protein as a reporter for the DNA damage-induced gene RAD54 in Saccharomyces cerevisiae. Yeast 13:1535-1545 (1997)

Walsh, Gary. Biopharmaceuticals: Biochemistry & Biotechnology. (2nd ed). John Wiley & Sons (2003)

Walsh, Gary, and Denis Headon. Protein Biotechnology. John Wiley & Sons. 1994

................
................

In order to avoid copyright disputes, this page is only a partial summary.

Google Online Preview   Download