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

Abstract

Background: 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 beta-lactoglobulin (BLG), a major cow's milk allergen, in the probiotic strain Lactobacillus casei BL23.

Results: 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 microg/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.

Conclusion: 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.

Figure 1

Expression cassettes for production and secretion of staphylococcal nuclease and/or bovine β-lactoglobulin. Nuc, staphylococcal nuclease, BLG, bovine β-lactoglobulin, P_nisA, nisin-inducible lactococcal promoter; RBS, ribosome binding site of usp45 gene; SP_Usp45, signal peptide of Usp45, LEISS, secretion-enhancer propeptide LEISSTCDA.

Figure 2

Dose-dependent nisin induction of staphylococcal nuclease production and secretion. L. casei BL23 strain was co-transformed with pSEC:Nuc and pNZ9520 plasmids. Protein extracts were analyzed by immunoblotting using rabbit polyclonal anti-Nuc antibodies as described in the Materials and Methods section. Arrows indicate positions of Nuc and its precursor form (pre-Nuc).

Figure 3

LEISS-Nuc-BLG production and secretion by L. casei BL23 and BL23 (int:nisRK) strains. Protein extracts were analyzed by immunoblotting using a specific anti-BLG monoclonal antibody for BL23 samples and rabbit polyclonal anti-Nuc antibodies for BL23 (int:nisRK) samples, as described in the Materials and Methods section. Arrows indicate the positions of LEISS-Nuc-BLG and precursor form.