Efficient production and secretion of bovine β-lactoglobulin by Lactobacillus casei

Stéphane Hazebrouck,Gérard Corthier,Philippe Langella,Vasco Azevedo,Jean-Michel Wal,Laetitia Pothelune

doi:10.1186/1475-2859-6-12

Abstract

BackgroundLactic 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.ResultsUsing 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.ConclusionBLG 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.

Highlights

Lactic acid bacteria (LAB) are attractive tools to deliver therapeutic molecules at the mucosal level
Nuc is efficiently secreted by L. casei We first investigated the ability of the BL23 strain to produce and secrete the reporter staphylococcal nuclease (Nuc, [15]) under the transcriptional control of the nisininducible promoter PnisA
We observed that fusion of the LEISS propeptide and Nuc, as initially described in L. lactis, improved both bovine βlactoglobulin (BLG) production and secretion in L. casei

Summary

Introduction

Lactic acid bacteria (LAB) are attractive tools to deliver therapeutic molecules at the mucosal level. Compared to recombinant lactococci, lactobacilli offer some advantages such as better survival in the digestive tract and immunomodulatory properties. We compared different strategies to optimize the production of bovine βlactoglobulin (BLG), a major cow's milk allergen, in the probiotic strain Lactobacillus casei BL23. Studies with recombinant lactobacilli are often impaired by the lower levels of antigen production compared to those obtained with L. lactis [12]. This is a major concern since mucosal immune response depends on the amount of antigen delivered by the bacterial vector [13]

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Conclusion