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Protein Purification by Ion Exchange Chromatography

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Textbook on Cloning, Expression and Purification of Recombinant Proteins

Abstract

Separation of similar biomolecules and proteins with little or no differences in molecular weight or without tags can be difficult with chromatographic techniques such as affinity or size exclusion. To circumvent this problem, distinct physicochemical properties of protein molecules have been harnessed for their separation. Since proteins carry overall electrical charges due to their chemical composition; ion exchange chromatography (IEX) uses this property to separate positively or negatively charged molecules via interaction with charged ion exchange resins as stationary media. Charged proteins bind to the resins in normal buffering conditions and can be gradually eluted with increasing salt concentration or by changing the pH of the mobile phase. Depending on the protein’s isoelectric point (pI) value, cation or anion exchange chromatography media can be used. If the pH environment of a protein is lower than its pI, it will carry a positive surface charge and strongly bind the cation exchange resins, while proteins with the negative surface charge will bind to the anion exchange counterpart. The purpose of this chapter on ion exchange chromatography is to describe its basic principle, protocols, applications in protein purification as well as provide troubleshooting tips.

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Acknowledgments

The authors thank Advanced Centre for Treatment, Research and Education in Cancer (ACTREC) for providing necessary infrastructure and resources for successful completion of the chapter. The authors acknowledge Ms. Chanda Baisane, Bose Lab for formatting the manuscript.

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Author notes

  1. Both the authors contributed equally to the work.

Authors and Affiliations

  1. Integrated Biophysics and Structural Biology Laboratory, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Mumbai, India

    Ayon Chakraborty

  2. Integrated Biophysics and Structural Biology Lab, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Mumbai, India

    Rashmi Puja & Kakoli Bose

  3. Homi Bhabha National Institute, BARC Training School Complex, Mumbai, India

    Rashmi Puja & Kakoli Bose

Authors
  1. Ayon Chakraborty
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  2. Rashmi Puja
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  3. Kakoli Bose
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Corresponding author

Correspondence to Kakoli Bose .

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Editors and Affiliations

  1. Integrated Biophysics and Structural Biology Lab, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Mumbai, India

    Kakoli Bose

Problems

Problems

Multiple Choice Questions

  1. 1.

    The capacity of the resin for ion exchange relies on:

    1. (a)

      The cumulative molecular mass of the resin

    2. (b)

      Length of the ion exchange resin

    3. (c)

      The total number of ion active groups

    4. (d)

      Solubility of the ion exchange resins

  2. 2.

    The concept of ion exchange chromatography is based on:

    1. (a)

      Electrostatic attraction

    2. (b)

      Electrical mobility of ionic species

    3. (c)

      Adsorption

    4. (d)

      Partition

  3. 3.

    In anion exchange chromatography:

    1. (a)

      The column contains negatively charged beads where positively charged proteins bind

    2. (b)

      The column contains positively charged beads where negatively charged proteins bind

    3. (c)

      The column contains both positive and negatively charged beads where proteins bind depending on their net charge

    4. (d)

      All of these

Subjective Questions

  1. 1.

    A protein has an isoelectric point (pI) of 5.2. What is the net charge on this protein in BICINE [(N, n-bis(2-hydroxyethyl)glycine] buffer (pH 8.5)? Explain.

  2. 2.

    A crude lysate sample comprising four proteins (1, 2, 3, and -galactosidase) is obtained by a protein biochemist. He wants to purify β-galactosidase using ion exchange chromatography. The respective isoelectric points of these proteins are enlisted below:

    Protein

    Isoelectric point (pI)

    1

    3.7

    2

    6.8

    3

    9.5

    β-Galactosidase

    5.3

He equilibrated an anion exchange column using a buffer of pH 5.0. (A) At this condition, which protein(s) from the lysate sample will bind to the column? (B) How the bound protein(s) can be eluted from the anion exchange column? He then recognized the fraction containing β-galactosidase from the anion exchange column and opted to purify it using a cation exchange column. (C) Explain how a cation exchange column may be used to separate β-galactosidase from any residual contaminated protein (s).

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Chakraborty, A., Puja, R., Bose, K. (2022). Protein Purification by Ion Exchange Chromatography. In: Bose, K. (eds) Textbook on Cloning, Expression and Purification of Recombinant Proteins. Springer, Singapore. https://doi.org/10.1007/978-981-16-4987-5_7

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