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Protein Purification by Affinity Chromatography

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Textbook on Cloning, Expression and Purification of Recombinant Proteins

Abstract

Affinity chromatography involves targeted purification of biological macromolecules from a crude mixture on the basis of highly specific interaction between the macromolecule and a tag protein or peptide. The interaction is typically reversible and purification is implemented by kee** one of the molecules (the affinity ligand or fusion tag) immobilized to the support matrix (containing respective binding resin for interaction with the tag) while its partner (the target protein) is in a mobile phase as part of the crude mixture. In this chapter we will be discussing recombinant protein purification using different affinity tags that are routinely used in a laboratory setup that include polyhistidine, GST (glutathione-S-transferase), maltose-binding protein (MBP) and Strep-tag. As affinity chromatography is a sophisticated purification method that requires significant expertise, the protocol and the problem-solving approaches described in this chapter will act as essential guides to the protein biochemists.

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Acknowledgements

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, ACTREC for formatting the manuscript.

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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

    Shubhankar Dutta

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

    Kakoli Bose

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

    Kakoli Bose

Authors
  1. Shubhankar Dutta
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  2. 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 specific biological interaction that is not used in affinity chromatography purification is:

    1. (a)

      Receptor-ligand

    2. (b)

      Antigen-antibody

    3. (c)

      Cations-anions

    4. (d)

      Enzyme-substrate

  2. 2.

    The first step of affinity chromatography purification process is:

    1. (a)

      Addition of affinity ligand into the matrix

    2. (b)

      Precipitation

    3. (c)

      Elution

    4. (d)

      Binding of the ligand with the tag

  3. 3.

    The property of an ideal affinity chromatography matrix is:

    1. (a)

      The matrix materials should be polymeric and organic

    2. (b)

      The matrix should be based on inorganic compounds

    3. (c)

      The matrix should be mechanically stable and exhibit good flow property

    4. (d)

      The matrix should form reversible but specific interaction with the affinity tag

Subjective questions

  1. 1.

    A 50 kDa His6×-tagged protein was adequately expressed in E. coli BL21 (DE3) host strain and subsequently purified using Ni-NTA column. Buffer conditions were as follows:

    Binding buffer: 50 mM NaH2PO4, 150 mM NaCl and 10 mM Imidazole, pH 7.4

    Elution buffer: 50 mM NaH2PO4, 150 mM NaCl and 100 mM imidazole, pH 7.4

    On purification, the protein co-elutes with chaperones and non-specific bands. In addition, white precipitates were observed in the eluted fractions. List a few strategies that can be employed to obtain better yield and purity of target protein.

  2. 2.

    To assess the interactions between protein A and protein B, an MBP pull-down assay was performed. Protein A (22 kDa) was tagged with MBP and considered the bait protein, whereas protein B (48 kDa) was kept untagged. The proteins were incubated in following buffer conditions:

    Binding buffer: 20 mM Tris–HCl, 200 mM NaCl, 1 mM EDTA, pH 7.4

    Elution buffer: 10 mM Maltose, 20 mM Tris–HCl, 200 mM NaCl, 1 mM EDTA, pH 7.4

    Post-elution, the pull-down eluates were subjected to SDS-PAGE analysis kee** only MBP as the control. The resultant gel analysis showed an unexpected band at 70 kDa which neither corresponds to protein A nor B. State a reason that can explain the band and also provide a strategy that can be implemented to avoid such bands.

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Dutta, S., Bose, K. (2022). Protein Purification by Affinity 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_6

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