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Cloning and Recombinant Protein Expression in Lactococcus lactis

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Advanced Methods in Structural Biology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2652))

Abstract

The Lactococcus lactis, a Gram-positive bacteria, is an ideal expression host for the overproduction of heterologous proteins in a properly folded and functional form. L. lactis has been identified as an efficient cell factory, generally recognized as safe (GRAS), has a long history of safe use in food production, and is known to have probiotic properties. Key desirable features of L. lactis include the following: (1) rapid growth to high cell densities, not requiring aeration which facilitates large-scale fermentation; (2) its Gram-positive nature precludes the presence of contaminating endotoxins; (3) the capacity to secrete stable recombinant protein into the growth medium with few proteases resulting in a properly folded, full-length protein; and (4) the availability of diverse expression vectors facilitating various cloning options. We have previously described production of several recombinant proteins with varying degrees of predicted structural complexities using the L. lactis pH-dependent P170 promoter. The purpose of this chapter is to provide a detailed protocol for facilitating wider application of L. lactis as a reliable platform for expression of heterologous recombinant proteins in soluble form. Here, we present details of the various steps involved such as cloning of the target gene in appropriate expression plasmid vector, determination of the expression levels of the heterologous protein, and initial purification of the expressed soluble recombinant protein of interest.

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Acknowledgments

The authors acknowledge the financial support granted by Danish Council for Strategic research (Grants 13127) and the Department of Biotechnology (DBT), Government of India (BT/IN/Denmark/13/SS/2013), European Union’s Horizon 2020 research and innovation program under grant agreement No. 733273, and the European and Developing Countries Clinical Trials Partnership (RIA2018SV-2311). SKS is supported by a DHR-NRI fellowship at ICMR. The authors also acknowledge funding from BIRAC-NBM, and the support of Director, ICMR-RMRC, Bhubaneswar, India.

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Authors and Affiliations

  1. Biotherapeutic and Vaccine Research Division, ICMR-Regional Medical Research Centre, Bhubaneswar, Odisha, India

    Susheel K. Singh & Subhash Singh

  2. Department for Congenital Disorders, Statens Serum Institut, Copenhagen, Denmark

    Mohammad Naghizadeh, Jordan Plieskatt & Michael Theisen

  3. Centre for Medical Parasitology at Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark

    Mohammad Naghizadeh & Michael Theisen

Authors
  1. Susheel K. Singh
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  2. Mohammad Naghizadeh
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  3. Jordan Plieskatt
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  4. Subhash Singh
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  5. Michael Theisen
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Editor information

Editors and Affiliations

  1. Universidade da Beira Interior, CICS-UBI, Covilhã, Portugal

    Ângela Sousa

  2. University of Beira Interior, Covilhã, Portugal

    Luis Passarinha

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Singh, S.K., Naghizadeh, M., Plieskatt, J., Singh, S., Theisen, M. (2023). Cloning and Recombinant Protein Expression in Lactococcus lactis. In: Sousa, Â., Passarinha, L. (eds) Advanced Methods in Structural Biology. Methods in Molecular Biology, vol 2652. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3147-8_1

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  • DOI: https://doi.org/10.1007/978-1-0716-3147-8_1

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3146-1

  • Online ISBN: 978-1-0716-3147-8

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