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Introduction to Recombinant Protein Purification

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Textbook on Cloning, Expression and Purification of Recombinant Proteins

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Abstract

Purified form of recombinant proteins is a prerequisite for undertaking in vitro biochemical and structural analyses of these macromolecules. Unfortunately, due to other proteins from the expression host, such as E. coli, the task of obtaining the desired protein from the heterologous system at highest purity and in sufficient quantity is arduous. With the increasing demand for recombinantly purified proteins both in basic and industrial research, over the last five decades, a plethora of research endeavors have been directed toward developing efficient protein purification techniques that would precisely amalgamate time and yield cost-effectively. However, it is extremely important to put careful forethought prior to developing a purification flow-scheme for a target protein to obtain the best possible output. This chapter outlines the general considerations to be undertaken while designing and streamlining these protocols with the help of recent advances in protein purification methodologies. It also provides an overview of the various chromatographic techniques that will be further elaborated in the succeeding chapters of this book.

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Acknowledgments

NS gratefully acknowledges financial support from the DBT-RA Program in Biotechnology and Life Sciences for completion of the manuscript. The authors acknowledge Ms. Chanda Baisane, Bose Lab, ACTREC for formatting the manuscript.

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Authors and Affiliations

  1. Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad, Haryana, India

    Nitu Singh

  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. Nitu Singh
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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.

    Protein purification techniques are based on the following properties except:

    1. (a)

      Solubility of the protein

    2. (b)

      Charge on the protein

    3. (c)

      Viscosity of the protein

    4. (d)

      Specific binding affinity of the protein

  2. 2.

    Salting-out refers to

    1. (a)

      Precipitation of proteins using ammonium sulfate

    2. (b)

      Precipitation of proteins using copper sulfate

    3. (c)

      Precipitation of proteins using sodium chloride

    4. (d)

      Both (a) and (c)

  3. 3.

    You find that your protein sample shows lots of additional bands of lower molecular weight apart from the desired protein. What can you do about this?

    1. (a)

      Add an additional purification step

    2. (b)

      Use a protease inhibitor during lysis and purification

    3. (c)

      Perform each step as quickly as possible, in a cold-room

    4. (d)

      All of the above

Subjective Questions

  1. 1.

    To estimate the molecular mass of an unknown protein, you decide to run a size exclusion chromatography. Next, you run a series of proteins with the known molecular mass and the unknown protein on a Sephadex G-200 column. Below are the elution volumes (Ve) for each protein. The measured void volume (V0) of the column is 36 mL. Using these values, calculate the molecular mass of the unknown protein.

    Table 5
    Full size table
  1. 2.

    To separate a mixture of proteins with different pI values, you try anion exchange chromatography using DEAE-cellulose column. For this, you first equilibrate your column with phosphate buffer, pH 6.5. Then you pass the following mixture of proteins, AS (pI – 4.6), BS (pI – 5.0), and CS (pI – 7.0). The proteins are eluted first with weak ionic strength buffer (50 mM NaCl, pH 6.5) and then in the same buffer but with increasing NaCl concentration. Explain what order will the proteins elute?

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Singh, N., Bose, K. (2022). Introduction to Recombinant Protein Purification. 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_5

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