Abstract
BackgroundStaphylococcal (or micrococcal) nuclease or thermonuclease (SNase or Nuc) is a naturally-secreted nucleic acid degrading enzyme that participates in Staphylococcus aureus spread in the infected host. Purified Nuc protein can be used as an exogenous reagent to clear cellular extracts and improve protein purification. Here, a recombinant form of Nuc was produced and secreted in a Gram-positive host, Lactococcus lactis, and purified from the culture medium.ResultsThe gene segment corresponding to the S. aureus nuclease without its signal peptide was cloned in an expression-secretion vector. It was then fused to a lactococcal sequence encoding a signal peptide, and expressed under the control of a lactococcal promoter that is inducible by zinc starvation. An L. lactis subsp cremoris model strain (MG1363) transformed with the resulting plasmid was grown in either of two media (GM17v and CDM) that are free of animal compounds, allowing GMP (Good Manufacturing Practice) production. Induction conditions (concentration of the metal chelator EDTA and timing of addition) in small-scale pH-regulated fermentors were optimized using LacMF (Lactis Multi-Fermentor), a home-made parallel fermentation control system able to monitor 12 reactors simultaneously. Large amounts of recombinant Nuc (rNuc) were produced and secreted in both media, and rNuc was purified from GM17v medium in a single-step procedure.ConclusionsIn L. lactis, rNuc production and secretion were optimal after induction by 0.5 mM EDTA in small scale (200 mL) GM17v exponential phase cultures (at an OD600 of 2), leading to a maximal protein yield of 210 mg per L of culture medium. Purified rNuc was highly active, displaying a specific activity of 2000 U/mg.
Highlights
Staphylococcal nuclease or thermonuclease (SNase or Nuc) is a naturally-secreted nucleic acid degrading enzyme that participates in Staphylococcus aureus spread in the infected host
L. lactis which is widely used as a starter in dairy industries, is an efficient cell factory for the production and secretion of proteins [1]. i) Proteins produced in this species, which is considered as safe by virtue of its longtime human consumption, should obtain the GRAS (Generally Recognized As Safe) status and be suitable for therapeutical or vaccine applications [1], in contrast to proteins produced in Escherichia coli from which endotoxin (LPS) has to be removed [2]. ii) In this Gram+ spe
Expression-secretion vector pGTP_FZ301 (Figure 1A) is an expression and secretion vector for L. lactis that is derived from pLB145 [13]. pGTP_FZ301 enables any ORF to be cloned as a translational fusion to the lactococcal sequence encoding Exp4 signal peptide [15,16], and the fusion and zitR constitute an operon under the control of the PZn promoter [12]
Summary
Staphylococcal (or micrococcal) nuclease or thermonuclease (SNase or Nuc) is a naturally-secreted nucleic acid degrading enzyme that participates in Staphylococcus aureus spread in the infected host. The lysostaphin from Staphylococcus simulans biovar staphylolyticus was successfully produced at the industrial scale (3000 L) in L. lactis using the NICE system Though, this naturally-secreted protein was produced as a recombinant signal peptide-free form that had to be purified from the lactococcal cell extract [8,19]
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