Heterologous expression and purification of the phage lysin-like bacteriocin LysL from Lactococcus lactis LAC460

Abstract The wild-type Lactococcus lactis strain LAC460 produces two bacteriocin-like phage lysins, LysL and LysP. This study aimed to produce and secrete LysL in various heterologous hosts and an in vitro cell-free expression system for further functional studies. Initially, the lysL gene from L. l...

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Bibliographic Details
Published in:FEMS microbiology letters 2024-01, Vol.371
Main Authors: Mokhtari, Samira, Saris, Per E J, Takala, Timo M
Format: Article
Language:English
Subjects:
Bacteriocins - biosynthesis
Bacteriocins - genetics
Bacteriocins - metabolism
Bacteriophages - genetics
Cloning, Molecular
Gene Expression
Lactococcus - genetics
Lactococcus - metabolism
Lactococcus - virology
Lactococcus lactis - genetics
Lactococcus lactis - metabolism
Lactococcus lactis - virology
Nisin - genetics
Nisin - metabolism
Nisin - pharmacology
Protein Sorting Signals - genetics
Research Letter
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Summary:Abstract The wild-type Lactococcus lactis strain LAC460 produces two bacteriocin-like phage lysins, LysL and LysP. This study aimed to produce and secrete LysL in various heterologous hosts and an in vitro cell-free expression system for further functional studies. Initially, the lysL gene from L. lactis LAC460 was cloned into Lactococcus cremoris NZ9000 and L. lactis N8 strains, with and without the usp45 signal sequence (SSusp45), under a nisin-inducible promoter. Active LysL was primarily produced intracellularly in recombinant L. lactis N8, with some secretion into the supernatant. Recombinant L. cremoris NZ9000 lysed upon nisin induction, indicating successful lysL expression. However, fusion with Usp45 signal peptide (SPUsp45–LysL) weakened LysL activity, likely due to incomplete signal peptide cleavage during secretion. Active LysL was also produced in vitro, and analysed in SDS-PAGE, giving a 42-kDa band. However, the yield of LysL protein was still low when produced from recombinant lactococci or by in vitro expression system. Therefore, His-tagged LysL was produced in Escherichia coli BL21(DE3). Western blot confirmed the intracellular production of about 44-kDa His-tagged LysL in E. coli. His-tagged active LysL was then purified by Ni-NTA affinity chromatography yielding sufficient 4.34 mg of protein to be used in future functional studies. Expressing the gene and purifying the protein of a novel bacterial enzyme.
ISSN:1574-6968
0378-1097
1574-6968
DOI:10.1093/femsle/fnae065