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Selection of Cloning and Expression Plasmid Vectors

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Textbook on Cloning, Expression and Purification of Recombinant Proteins

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Abstract

Purification of proteins using recombinant DNA technology is crucial for studying its structure, function, and interactions with other ligands/macromolecules as well as for therapeutic purposes. Thus, the need for purified proteins has been increasing day by day both in basic research labs and at the industrial level. Major developments in the field of recombinant DNA technology have been focused on improving the process of protein purification. However, the very first step in this process is cloning and expression of isolated protein coding genes through engineered plasmid molecules (self-replicating extrachromosomal circular DNA molecules) called vectors. Therefore, with the increasing demand for purified proteins, the need for more efficient and robust vectors was perceived. Today, a wide array of vectors for both cloning and expressing the gene of interest are being engineered that suit almost every requirement of the researcher. However, the challenge lies in choosing the correct vector for a specific requirement, and hence a thorough knowledge of all the available vectors used for different purposes becomes imperative. This chapter aims at guiding the researchers toward choosing appropriate vectors to cater to their various cloning requirements. It also provides troubleshooting tips and discusses latest advancements in this technology.

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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 and Mrs Snehal Pandav Mudrale, Bose Lab, ACTREC for critical inputs and formatting of the manuscript.

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Authors and Affiliations

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

    Rucha Kulkarni, Roshnee Bose & Kakoli Bose

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

    Rucha Kulkarni & Kakoli Bose

Authors
  1. Rucha Kulkarni
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  2. Roshnee Bose
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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 site on an expression plasmid where transcription factors bind is known as:

    1. (a)

      Ori

    2. (b)

      Promoter

    3. (c)

      Polylinker

    4. (d)

      rop

  2. 2.

    The most suitable vector for constructing genomic libraries would be:

    1. (a)

      Cosmids

    2. (b)

      λ replacement vectors

    3. (c)

      BAC

    4. (d)

      pET vectors

  3. 3.

    The protein that binds to the pLac promoter to prevent the expression of lacZ gene is known as:

    1. (a)

      Lactose

    2. (b)

      IPTG

    3. (c)

      X- gal

    4. (d)

      Repressor

Subjective Questions

  1. 1.

    A gene encoding protein X was cloned into a plasmid containing 6×-His tag. After induction of gene expression, the protein was found to be expressed. When the protein was purified using an appropriate resin, it was found to be unstable and insoluble. Suggest a way in which such a protein can be stabilized and purified.

  2. 2.

    A gene 50–60 kB in size needs to be cloned in an appropriate vector for further study. Which cloning vector would be suitable for cloning such a moderately large fragment without loss of the gene and at the same time ensuring efficient host transformation?

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Kulkarni, R., Bose, R., Bose, K. (2022). Selection of Cloning and Expression Plasmid Vectors. 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_3

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