Abstract
The Gram-positive bacterium Lactococcus lactis is an ideal expression host for the overproduction of heterologous proteins in a functional form. L. lactis has recently been identified as an efficient Gram-positive cell factory for the production of recombinant proteins and the safety of this production system has been confirmed in multiple clinical trials. Key desirable features of L. lactis include its generally recognized as safe (GRAS) status, long history of safe use in food production, probiotic properties, absence of endotoxins, capacity to secrete stable recombinant protein to the growth medium, the presence of few proteases, and a diverse selection of cloning and inducible expression vectors. Growth of lactococci is rapid, proceeds to high cell densities, and does not require aeration, which facilitates large-scale fermentation. We have previously described the production of several Plasmodium falciparum antigens with varying degrees of predicted structural complexities, those which are considered difficult-to-produce proteins by using L. lactis pH-dependent inducible promoter (P170). The purpose of this chapter is to provide a detailed protocol for the expression of difficult-to-produce proteins, mainly high cysteine-rich proteins, in the soluble form in L. lactis from cloning of the target gene to the determination of expression levels and purification.
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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, the European and Developing Countries Clinical Trials Partnership (RIA2018SV-2311) and by PATH’s Malaria Vaccine Initiative under Grant OPP1108403 from the Bill & Melinda Gates Foundation and in part by Grant NNF14CC0001.
We also thank Michael Theisen (Statens Serum Institut, Copenhagen, Denmark) for kindly allowing us to illustrate this chapter with the figures on the expression plasmid for L. lactis system and technical review of the prepared manuscript, and valuable feedback.
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Singh, S.K., Singh, V. (2022). Method for Production of Cysteine-Rich Proteins in Lactococcus lactis Expression System. In: Garcia Fruitós, E., ArÃs Giralt, A. (eds) Insoluble Proteins. Methods in Molecular Biology, vol 2406. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1859-2_11
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DOI: https://doi.org/10.1007/978-1-0716-1859-2_11
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