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Technical Bulletin

Protein Purification Techniques


Vol. 4. Metal-Chelate Affinity Chromatography

Introduction β-mercaptoethanol can be used with nickel chelated matrices,


Metal-Chelate Affinity Chromatography (MCAC), also known although lower concentrations should be used whenever
as Immobilized Metal Affinity Chromatography (IMAC), was possible.
first successfully demonstrated in 1975 by Porath and
collaborators for human serum proteins. MCAC utilizes metal For most MCAC applications, including His-Tagged protein
ions bound to solid matrices that bind to the exposed imidazole purification, the pH is critical for initial binding and subsequent
and thiol groups of proteins. MCAC commonly utilizes zinc elution of bound proteins. Typically, binding occurs at neutral
(Zn2+), nickel (Ni2+) or copper (Cu2+) to form stable complexes or slightly alkali pH (6.5 - 8.0), whereas elution generally
with histidine, tryptophan and cysteine residues within proteins. occurs under acidic environments (< 6.0). It is important to
Cadmium, mercury, cobalt, calcium or iron ions can also be utilize compatible buffer systems that maintain their pH
used, but are less common. The affinity is not specific, but accurately at all temperatures experienced during the binding
MCAC can preferentially isolate metal-ion-binding proteins. and elution of the proteins.
Once bound, the proteins can be eluted via pH or imidazole
gradients. Typically, a pH gradient is employed to elute the proteins of
interest, however bound proteins can also be eluted with an
Key Parameters for the Operation of MCAC imidazole gradient at a stable pH. This method provides much
Initially, the solid matrix must be charged with the metal salt simpler operation, but may not be as effective as a pH gradient.
solution. The best choice of immobilized metal is not always For proteins with a high affinity for the metal ions, a buffer
predictable and it is typically determined by trial and error. with a pH less than 6.0 and a chelating agent (e.g. EDTA or
Generally, copper binds proteins with greater affinity than EGTA) can be used to strip the matrix of all bound metal, and
zinc. However, zinc’s lower affinity makes it suitable for certain subsequently the bound proteins. Care must be taken when
applications. For the purification of histidine-rich proteins, or performing this method, as it is possible the proteins may
those proteins with an exposed histidine tail (His-Tagged), precipitate due to the chelating agents.
nickel is the preferred choice.
MCAC Purification of Proteins
If the protein of interest does not bind to the matrix, either From Cell Culture Media
the matrix is not properly charged with metal ions, the protein As with most protein chromatography, it is typically
lacks sufficient imidazole (i.e. histidine residues) and thiol recommended that the spent (nutrient depleted) cell culture
groups or the imidazole and thiol groups are inaccessible to media be removed via a buffer exchange step (e.g. dialysis,
the immobilized metal ions. Chelating agents, such as tangential flow, etc.). Although this step can ensure the
ethylenediaminetetracetic acid (EDTA) and ethylene glycol- removal of most media components, it is not always desirable
bis(β-aminoethyl ether) N, N, N’, N’,-tetraacetic acid (EGTA), due to the time and equipment involved. MCAC can
must be excluded from all solutions because they will strip sometimes be used with spent cell culture media without a
the metal ions from the matrix. Likewise, strong reducing buffer exchange step, however, some experimentation may
agents such as dithiothreitol and β-mercaptoethanol should be required to find the optimal parameters.
also be avoided because they can reduce the metal ions and
greatly lower MCAC efficiency. Up to 10 mM

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SAFC Biosciences, Inc. SAFC Biosciences Ltd. SAFC Biosciences Pty. Ltd.
13804 W. 107th Street Smeaton Road, West Portway 18-20 Export Drive
Lenexa, Kansas 66215 Andover, Hampshire SP10 3LF Brooklyn, Victoria 3025
USA
Phone
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Most serum-free media contain a surfactant, Pluronic® F68, References
to protect the cells from the shear forces common in 1. Porath, J., Carlson, J., Olsson, I., and Belfrage, G., Metal Chelate
suspension cultures. It is likely that Pluronic® F68 will not Affinity Chromatography: A New Approach to Protein Fractionation,
affect the initial use of a new column, but over time it may Nature (1975) 258:598.
lower the binding efficiency of the column unless fully removed 2. Asenjo, Juan A., Separation Processes in Biotechnology, New York:
by adequate washing of the solid matrix. If necessary, Pluronic® Marcel Dekker, 1990.
F68 can be removed by buffer exchange without damaging 3. Janson, Jan-Christer and Lars Ryden, Protein Purification, New
the proteins. York: VCH Publishers, Inc., 1989.
4. Harrison, Roger G., Protein Purification Process Engineering, New
Serum-free media may also contain a hydrolysate to aid in cell York: Marcel Dekker, 1993.
growth. Hydrolysates are peptide fragments created by 5. Deutscher, Murray P., Methods in Enzymology, vol. 182, Guide to
hydrolysis of source proteins, typically from plants or yeast. Protein Purification, New York: Academic Press, 1990.
Hydrolysates are very common in serum-free media, but they
can prove troublesome during protein purification. They can
plug columns and block the binding sites of the column’s
matrix. Higher flow rates and the use of prefiltration can
minimize their effect, however it cannot be predicted how the
hydrolysates will react in your system. As with Pluronic® F68,
hydrolysates can be removed by buffer exchange.

Most spent cell culture media may require alterations to


perform adequately in MCAC. After use, mammalian cell
culture media may have a pH less than 6.0 (insect cell culture
media; less than 5.5). As previously mentioned, the optimal
pH for MCAC binding is near neutral. Spent media will have
to be adjusted to the proper pH. Most spent mammalian cell
culture media can be adjusted to a pH of 7.0 - 8.0 with a
sodium hydroxide solution. Unfortunately, serum-free insect
cell culture media may precipitate when the pH is raised
above 7.1, therefore, it should be adjusted to a pH of
6.6 - 6.9 with a potassium hydroxide solution.

In addition to an optimal pH, MCAC typically requires


0.5 - 1.0 M salt (sodium chloride or potassium chloride) to
minimize non-specific binding. Most cell culture media have
50 - 200 mM. Additional salt (typically an additional 0.5 M
sodium chloride) should be added to the spent media to limit
non-specific binding. It may also be helpful to slightly dilute
the medium to aid in the binding and minimize the effects
of components such as Pluronic® F68 and the hydrolysates
common in serum-free media. Warranty, Limitation of Remedies
SAFC Biosciences warrants to the purchaser for a period of one year from date of delivery that this product conforms to
its specifications. Other terms and conditions of this warranty are contained in SAFC Biosciences’ written warranty, a
Metal-Chelate Affinity Chromatography and protein purification copy of which is available upon request. ALL OTHER WARRANTIES, EXPRESSED OR IMPLIED, INCLUDING THE IMPLIED
WARRANTY OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE, ARE EXCLUDED. In no case will SAFC
in general are very protein specific and require a great deal Biosciences be liable for any special, incidental, or consequential damages arising out of this product or the use of this
product by the customer or any third party based upon breach of warranty, breach of contract, negligence, strict tort,
of optimization. or any other legal theory. SAFC Biosciences expressly disclaims any warranty against claims by any third party by way of
infringement or the like. THIS PRODUCT IS INTENDED FOR PURPOSES DESCRIBED ONLY AND IS NOT INTENDED FOR
ANY HUMAN OR THERAPEUTIC USE.

For further assistance, please contact our Technical Services Additional Terms and Conditions are contained in the product Catalog, a copy of which is available upon request.

department.
Pluronic® is a registered trademark of BASF Corporation.

© 2006 SAFC Biosciences, Inc.

Issued April 2006 T045


0103 1205

United States Europe Asia Pacific


SAFC Biosciences, Inc. SAFC Biosciences Ltd. SAFC Biosciences Pty. Ltd.
13804 W. 107th Street Smeaton Road, West Portway 18-20 Export Drive
Lenexa, Kansas 66215 Andover, Hampshire SP10 3LF Brooklyn, Victoria 3025
USA UNITED KINGDOM AUSTRALIA

www.safcbiosciences.com Phone
Toll free-USA
Fax
+1 913-469-5580
1 800-255-6032
+1 913-469-5584
Phone
Fax
E-mail
+44 (0)1264-333311
+44 (0)1264-332412
info-eu@sial.com
Phone
Toll free-AUS
Fax
+61 (0)3-9362-4500
1 800-200-404
+61 (0)3-9315-1656
E-mail info-na@sial.com E-mail info-ap@sial.com