Heterologous protein secretion in Lactococcus lactis: a novel antigen delivery system

Lactic acid bacteria (LAB) are Gram-positive bacteria and are generally reg arded as safe (GRAS) organisms. Therefore, LAB could be used for heterologo us protein secretion and they are good potential candidates as antigen deli very vehicles. To develop such live vaccines, a better control of protein s ecretion is required. We developed an efficient secretion system in the mod el LAB, Lactococcus lactis. Staphylococcal nuclease (Nuc) was used as the r eporter protein. We first observed that the quantity of secreted Nuc correl ated with the copy number of the cloning vector. The nuc gene was cloned on a high-copy number cloning vector and no perturbation of the metabolism of the secreting strain was observed. Replacement of nuc native promoter by a strong lactococcal one led to a significant increase of nuc expression. Se cretion efficiency (SE) of Nuc in L. lactis was low, i.e., only 60% of the synthesized Nuc was secreted. Insertion of a synthetic propeptide between t he signal peptide and the mature moiety of Nuc increased the SE of Nuc. On the basis of these results, we developed a secretion system and we applied it to the construction of an L. lactis strain which secretes a bovine coron avirus (BCV) epitope-protein fusion (BCV-Nuc). BCV-Nuc was recognized by bo th anti-BCV and anti-Nuc antibodies. Secretion of this antigenic fusion is the first step towards the development of a novel antigen delivery system b ased on LAB-secreting strains.