Production and purification of staphylococcal nuclease in Lactococcus lactis using a new expression-secretion system and a pH-regulated mini-reactor.
BACKGROUND
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. 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.


RESULTS
The 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.


CONCLUSIONS
In 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 OD(600) 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.
Trémillon
Nicolas
N
GTP-Technology, Immeuble Biostep, BP 48184, 31681 Labège Cedex, France.
Issaly
Nicolas
N
Mozo
Julien
J
Duvignau
Thomas
T
Ginisty
Hervé
H
Devic
Eric
E
Poquet
Isabelle
I
D019149:Bioreactors; D003001:Cloning, Molecular / Q000379:methods*; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D013294:Lactococcus lactis / Q000235:genetics*; D008836:Micrococcal Nuclease / Q000096:biosynthesis* / Q000302:isolation & purification; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013211:Staphylococcus aureus / Q000201:enzymology
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D008836:Micrococcal Nuclease