Molecular Techniques and Methods

Protein Purification by Ion Exchange Chromatography

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Ion-exchange chromatography separates proteins based on molecular charge. Protein mixtures are applied to in oppositely charged, chromatographic matrix and the various proteins are bound by reversible, electrostatic interactions. The adsorbed proteins are eluted in order of least to most strongly bound molecules (by increasing the ionic strength or varying the pH of the elution buffer), collected as individual chromatographic fractions, and analyzed separatetely.




MATERIALS AND SOLUTIONS

  • Factors to be Considered for Selection of an Ion-Exhcange Gel Matrix and Column

    Proteins carry both negatively and positively charged groups. The net charge of a protein is dependent on pH. At its isoelectric point (pI), the net charge of a protein is zero and no binding to any type of ion-exchange gel matrix will occur. The following factors should be considered.

    (1) pH Range Where the Protein of Interest is Stable.
    If the protein is most stable at pH's above its pI, then an anion-exchange gel matrix should be used. If it is most stable below its pI, then a cation-exchange gel matrix should be used.

    (2) Molecular Size of the Protein being Separated.
    The porosity of an ion-exchange matrix affects the binding capacity of a gel matrix. For proteins of molecular weight 10,000 to 100,000, DEAE-Sephacel and DEAE-Sepharose are good choices. For larger proteins, Sephadex A-25 or C-25, which have the highest charge density at the surface of the gel, are appropriate.

    (3) Operating Pressue.
    For nonrigid gels (e.g., Sephacel or Sepharose), operating pressures should not exceed the manufacturer defined limits. For rigid gels (e.g., Analytical grade (AG), Bio-Rex MSZ, and Dowex commercial grade ion-exchange resins from Bio-Rad), packing can be carried out at higher flow rates using a peristaltic pump.

    (4) Choice of Column Size depends on the Binding Capacity of the Ion-Exchange Gel Matrix.
    The column diameters most frequently used are 1, 2, and 2.5 cm. Reservoir design, direction of column flow, and whether or not a peristaltic pump should be employed are arbitrary.
    Separating columns are usually <20 cm in length. If the protein mixture is exceedingly complex, a longer column should be used.



  • Various Ion-Exchange Matrices are Available Commercially.
    These gels differ in chemical nature of the gel matrix, type and degree of cross-linking, particle size, type of exchanger (anionic vs. cationic-as well as level of substitution of charged groups), chemical stability, and physical stability.

  • Commonly Used Anion-Exchange Matrices for Protein Seperation

    Cellulose Matrix
    Exchange Type
    Aminoethyl-Cellulose
    Weak
    Diethylaminoethyl (DEAE)-Cellulose
    Weak
    Benzyl DEAE-Cellulose
    Weak
    Polyethylenimine (PEI)-Cellulose
    Weak
    Diethyl-[2-hydroxypropyl]-aminoehtyl (DAE)-Cellulose
    Strong
    Triethylaminoethyl (TEAE)-Cellulose
    Weak
    Diethylaminoethyl (DEAE)-Sephacel
    Weak


    Polystyrene Matrix
    Exchange Type
    Analytical Grade (AG)-1
    Strong
    Analytical Grade (AG)-2
    Strong
    Bio-Rex 5
    Intermediate
    AG 3-X4A
    Strong
    Bio-Rex MSZ 1-X8
    Strong


    Sephadex Matrix
    Exchange Type
    Diethylaminoethyl (DEAE)-Sephadex
    Weak
    Diethyl-[2-hydroxypropyl]-aminoehtyl (DAE)-Sephadex
    Strong


    Sepharose Matrix
    Exchange Type
    Diethylaminoethyl (DEAE)-Sepharose CL-6B
    Weak
    Diethylaminoethyl (DEAE)-Sepharose (fast flow)
    Weak
    Quaternary amine (Q)-Sepharose (fast flow)
    Strong



  • Commonly Used Cation-Exchange Matrices for Protein Seperation

    Cellulose Matrix
    Exchange Type
    Carboxymethyl (CM)-Cellulose
    Weak


    Polystyrene Matrix
    Exchange Type
    AG 50W
    Strong
    Bio-Rex 70
    Weak


    Sephadex Matrix
    Exchange Type
    Carboxymethyl (CM)-Sephadex
    Weak
    Sulphopropyl (SP)-Sephadex
    Strong


    Sepharose Matrix
    Exchange Type
    Carboxymethyl (CM)-Sepharose CL-6B
    Weak
    Carboxymethyl (CM)-Sepharose (fast flow)
    Weak
    Sulphonate (S)-Sepharose (fast flow)
    Strong



  • Commonly Used Anion-Exchange Buffers for Protein Separation

    pH Gradient
    Buffers
    2.3 to 3.5
    Pyridine/ Formic acid
    3.0 to 6.0
    Pyridine/ Acetic acid
    7.0 to 8.5
    Ammonia/ Formic acid
    8.5 to 10.0
    Ammonia/ Acetic acid
    7.0 to 12.0
    Triethylamine/ CO2
    7.6 to 8.6
    0.05 M Tris-HCl + 1 M NaCl


  • Commonly Used Cation-Exchange Buffers for Protein Separation

    pH
    Buffers
    2.0
    Formic acid
    3.5
    0.06 M Sodium formate + 0.95 M NaCl
    6.0
    0.05 MES + 1 M NaCl

    If a chaotropic agent (e.g., urea), organic solvent (e.g., alcohol or acetonitrile), or detergent (e.g., nonionic forms) is required to solubilize the protein of interest, it may be added to the gradient buffers. It is important to demonstrate that the buffer salts do not precipitate in the presence of these additives.




    PROCEDURES

    Preparation of Ion-Exchange Gel Matrix and Column Packing

    1. Swell the ion-exchange gel matrix in an appropriate ion-exchange buffer.
  • Allow to swell completely.

    2. After allowing the gel to settle, aspirate or decant the fine gel particles which have not settled to the gel bed.

    3. Resuspend the settled gel in an equal volume of ion-exchange buffer to form a thick suspension, pour the slurry into a filtration flask.
  • Degas the gel in order to remove trapped air.

    4. The volume of swelling buffer and the swelling time required for a given gel depends on the gel type. The dry gel should be gradually added to the buffer, while gently stirring the suspension with a glass rod.
    Note: Do not stir with a magnetic stirrer, since the gel beads will be pulverized.

    5. Suspend the gel in twice the approximate bed volume (i.e., milliliters of swollen gel).
  • Gels may be rapidly swollen by heating the slurry at 90oC for 5 hours, using a water bath to control the temperature. Swelling at room temperature can be considerably slower, especially for large pore gels.

    6. If a ion-exchange gel matrix is purchased preswollen, it should be washed with excess buffer on a Buchner funnel to remove antimicrobial agents contained in the storage buffer. Resuspend the settled gel in ion-exchange buffer, degas the suspension.

    7. Mount the column vertically on a laboratory stand. A carpenter's level should be used to determine that the column is vertical.

    8. Using a syringe, inject ion-exchange buffer into the column outlet tubing until the empty column is filled with buffer to just above the bed support screen. Leave the syringe in place to block the end of the outlet tubing.
  • (This procedure removes trapped air from below the support screen.)

    9. Pour an appropriate volume of the gel suspension in order to fill completely the column to the required column bed height.
  • The gel suspension should be poured onto a glass rod whose end touches the inner column wall. This will result in a smooth flow of the gel suspension without unnecessary turbulence and introduction of air.

    10. Since the volume of the slurry usually exceeds the desired column bed volume, it is convenient to use a gel reservoir or column extension to hold the excess slurry.

    11. After the column is packed to the desired bed height, carefully pipet a 1 cm layer of buffer onto the top of the gel. After completely filling the inlet tubing and end fitting with buffer, connect the end fitting to the column.

    12. Before removing the syringe from the outlet tubing, adjust the buffer reservoir to provide an appropriate operating pressure or use a peristaltic pump to control the flow rate.

  • For nonrigid gels (e.g., Sephacel or Sepharose), operating pressures should not exceed the manufacturer defined limits.
  • For rigid gels (e.g., Analytical grade (AG), Bio-Rex MSZ, and Dowex commercial grade ion-exchange resins from Bio-Rad), packing can be carried out at higher flow rates using a peristaltic pump.

    13. Remove the syringe from the column outlet tubing and start the flow. Wash the column with 2 to 3 bed volumes of buffer in order to pack the bed and to equilibrate the column with buffer. A slightly higher flow rate can be used for packing than will be used for chromatographic separation.

    14. Close the outlet tubing. Inspect the packed bed, illuminating from behind by a flashlight to detect cracks or trapped air in the column bed.



    Applying Sample and Running the Gradient

    15. Dissolve the sample containing a mixture of proteins in the starting gradient buffer.

    16. Close the outlet tubing and remove most of the buffer from above the column bed by aspiration. Open the outlet tubing and allow the remaining buffer to penetrate into the gel, but do not let center of gel become dry. Close the outlet tubing.

    17. Using a Pasteur pipet, apply a sample of a protein. Any volume may be applied so long as the ionic strength of the solution does not exceed the ionic strength of the starting buffer.

    18. Open the column outlet tubing and allow sample to penetrate into the bed.

    19. Wash the remaining sample from the column wall by applying small amounts of buffer from a Pasteur pipet.

    20. After the sample is applied, carefully pipet a 1 cm layer of starting buffer onto the top of the gel, attach the gradient mixer to the meet tubing and, after completely filling the inlet tubing and end filling with starting buffer, connect the end fitting to the column.

    21. Open the two valves of the gradient mixer. Open outlet tubing. Allow gradient elution to proceed at an appropriate hydrostatic pressure.

    22. Collect the column effluent using an automated fraction collector.
  • It is convenient to collect 100 fractions each containing a volume equivalent to 1 % of the total gradient volume. Measure the A280 of each fraction.

  • Although column fractions can be collected by time or by drop number, it is preferable to collect fractions by drop number in order to avoid variations inflow rate giving rise to different fraction volumes. This variation is observed with ion-exchange chromatography, since at higher salt concentrations the chromatographic matrix contracts progressively leading to higher flow rates.




    NOTES

  • Conditions for adsorption of a protein of interest to an ion-exchange matrix must be found empirically. As a rule of thumb an ion exchanger can be selected for a protein of interest on the basis of its pI.

    pI of Protein
    Ion-Exchange Matrix
    8
    Cation-Exchanger, pH<7
    7
    Cation-Exchanger, pH<6
    5.5
    Anion-Exchanger, pH>6.5

  • If the pI is not known, the appropriate matrix and chromatographic elution conditions can be determined as in the second support protocol. If the pH required for a given separation is < 6 or > 9, then a strong ion exchanger should be used. Otherwise a weak exchanger should be used, because of higher binding capacity at neutral pH.




    KIT INFORMATION




    REFERENCES

  • Himmelhoch, SR (1971) Chromatography of proteins on ion-exchange adsorbents. Meth. Enzymol. 22:273-286.

  • Peterson, EA, Sober, HA (1956) Chromatography of proteins. 1. Cellulose ion-exchange adsorbents. J. Am. Chem. Soc. 78:751-758.

  • Scopes, RK (1982) Ion exchangers-principles, properties and uses. In "Protein Purification: Principles and Practice", pp75-101. Springer-Verlag, New York.


  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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