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

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.