TY - CHAP

T1 - Cloning and recombinant protein expression in Lactococcus lactis

AU - Singh, Susheel K.

AU - Naghizadeh, Mohammad

AU - Plieskatt, Jordan

AU - Singh, Subhash

AU - Theisen, Michael

N1 - Publisher Copyright: © 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

PY - 2023

Y1 - 2023

N2 - 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.

AB - 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.

KW - Cloning

KW - Expression

KW - Lactococcus lactis

KW - Protein secretion

KW - Purification

KW - Recombinant protein

U2 - 10.1007/978-1-0716-3147-8_1

DO - 10.1007/978-1-0716-3147-8_1

M3 - Book chapter

C2 - 37093467

AN - SCOPUS:85153687535

SN - 978-1-0716-3146-1

SN - 978-1-0716-3149-2

T3 - Methods in Molecular Biology

SP - 3

EP - 20

BT - Advanced Methods in Structural Biology

A2 - Sousa, Ângela

A2 - Passarinha, Luis

PB - Humana Press

ER -