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

Microbial Cell Factories

Research

Hervé Ginisty¹  Thomas Duvignau¹  Eric Devic¹  Julien Mozo¹  Nicolas Issaly¹  Nicolas Trémillon²  Isabelle Poquet³ 

[1] GTP-Technology, Immeuble Biostep, BP 48184, 31681, Labège Cedex, France;GTP-Technology, Immeuble Biostep, BP 48184, 31681, Labège Cedex, France;INRAUMR1319 Micalis (Microbiologie de l'Alimentation au service de la Santé), Domaine de Vilvert, Bâtiment 222, F-78352, Jouy-en-Josas cedex, France;INRAUMR1319 Micalis (Microbiologie de l'Alimentation au service de la Santé), Domaine de Vilvert, Bâtiment 222, F-78352, Jouy-en-Josas cedex, France;

关键词: Heterologous Protein;    Induction Condition;    Chemically Define Medium;    Staphylococcal Nuclease;    Uninduced Cell;   

DOI  :  10.1186/1475-2859-9-37

 received in 2009-06-03, accepted in 2010-05-21,  发布年份 2010

来源: Springer

【 摘 要 】

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.

【 授权许可】

Unknown   
© Trémillon et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.