Efficient production and secretion of bovine β-lactoglobulin by Lactobacillus casei
Stéphane Hazebrouck1, Laetitia Pothelune1, Vasco Azevedo3 , Gérard Corthier2 , Jean-Michel Wal1 and Philippe Langella2
1INRA, Unité d’Immuno-Allergie Alimentaire, CEA de Saclay, 91191 Gif-sur-Yvette, France
2INRA, Unité d’Ecologie et Physiologie du Système Digestif, 78352 Jouy-en-Josas cedex, France
3Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte – MG, Brasil
Lactic acid bacteria (LAB) are attractive tools to deliver therapeutic molecules at the mucosal level. The model LAB Lactococcus lactis has been intensively used to produce and deliver such heterologous proteins. However, compared to recombinant lactococci, lactobacilli offer some advantages such as better survival in the digestive tract and immunomodulatory properties. Here, we compared different strategies to optimize the production of bovine β-lactoglobulin (BLG), a major cow’s milk allergen, in the probiotic strain Lactobacillus casei BL23.
Using a nisin-inducible plasmid system, we first showed that L. casei BL23 strain could efficiently secrete a reporter protein, the staphylococcal nuclease (Nuc), with the lactococcal signal peptide SPUsp45 fused to its N-terminus. The fusion of SPUsp45 failed to drive BLG secretion but led to a 10-fold increase of intracellular BLG production. Secretion was significantly improved when the synthetic propeptide LEISSTCDA (hereafter called LEISS) was added to the N-terminus of the mature moiety of BLG. Secretion rate of LEISS-BLG was 6-fold higher than that of BLG alone while intracellular production reached then about 1 mg/L of culture. The highest yield of secretion was obtained by using Nuc as carrier protein. Insertion of Nuc between LEISS and BLG resulted in a 20-fold increase in BLG secretion, up to 27 μg/L of culture. Furthermore, the lactococcal nisRK regulatory genes were integrated into the BL23 chromosome. The nisRK insertion allowed a decrease of BLG synthesis in uninduced cultures while BLG production increased by 50% after nisin induction. Moreover, modification of the induction protocol led to increase the proportion of soluble BLG to around 74% of the total BLG production.
BLG production and secretion in L. casei were significantly improved by fusions to a propeptide enhancer and a carrier protein. The resulting recombinant strains will be further tested for their ability to modulate the immune response against BLG via mucosal delivery in a cow’s milk allergy model in mice.
Microbial Cell Factories 2007, 6:12. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.