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Applied and Environmental Microbiology, June 2002, p. 3141-3146, Vol. 68, No. 6
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.6.3141-3146.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Controlled Production of Stable Heterologous Proteins in Lactococcus lactis

A. Miyoshi,1,2 I. Poquet,1 V. Azevedo,2 J. Commissaire,1 L. Bermudez-Humaran,1,3 E. Domakova,1 Y. Le Loir,1 S. C. Oliveira,2 A. Gruss,1 and P. Langella1*

Unité de Recherches Laitières et de Génétique Appliquée, INRA, Domaine de Vilvert, 78352 Jouy en Josas cedex, France,1 Institute of Biological Sciences, Federal University of Minas Gerais (UFMG-ICB), Belo Horizonte, Minas Gerais, Brazil,2 Laboratorio de Inmunología y Virología, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, San Nicolas de los Garza, Nuevo León, Mexico3

Received 5 September 2001/ Accepted 4 March 2002

The use of Lactococcus lactis (the most extensively characterized lactic acid bacterium) as a delivery organism for heterologous proteins is, in some cases, limited by low production levels and poor-quality products due to surface proteolysis. In this study, we combined in one L. lactis strain use of the nisin-inducible promoter PnisA and inactivation of the extracellular housekeeping protease HtrA. The ability of the mutant strain, designated htrA-NZ9000, to produce high levels of stable proteins was confirmed by using the staphylococcal nuclease (Nuc) and the following four heterologous proteins fused or not fused to Nuc that were initially unstable in wild-type L. lactis strains: (i) Staphylococcus hyicus lipase, (ii) the bovine rotavirus antigen nonstructural protein 4, (iii) human papillomavirus antigen E7, and (iv) Brucella abortus antigen L7/L12. In all cases, protein degradation was significantly lower in strain htrA-NZ9000, demonstrating the usefulness of this strain for stable heterologous protein production.

* Corresponding author. Mailing address: Unité de Recherches Laitières et de Génétique Appliquée, INRA, Domaine de Vilvert, 78352 Jouy en Josas cedex, France. Phone: 33 1 34 65 20 83. Fax: 33 1 34 65 20 65. E-mail: langella{at}jouy.inra.fr.

Applied and Environmental Microbiology, June 2002, p. 3141-3146, Vol. 68, No. 6
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.6.3141-3146.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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