Cloning and recombinant protein expression in Lactococcus lactis

Susheel K. Singh*, Mohammad Naghizadeh, Jordan Plieskatt, Subhash Singh, Michael Theisen

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-review

3 Citations (Scopus)

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.

Original languageEnglish
Title of host publicationAdvanced Methods in Structural Biology
EditorsÂngela Sousa, Luis Passarinha
Number of pages18
PublisherHumana Press
Publication date2023
Pages3-20
ISBN (Print)978-1-0716-3146-1, 978-1-0716-3149-2
ISBN (Electronic)978-1-0716-3147-8
DOIs
Publication statusPublished - 2023
SeriesMethods in Molecular Biology
Volume2652
ISSN1064-3745

Bibliographical note

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

Keywords

  • Cloning
  • Expression
  • Lactococcus lactis
  • Protein secretion
  • Purification
  • Recombinant protein

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