FIG. 1.
Secretion of Blg41-60::Nuc fusion protein in L. lactis. (A) Expression cassettes for production and export of Nuc and fusion protein Blg41-60::Nuc. Schematic structures of Nuc and the fusion protein carried by the indicated plasmids are shown. For details of plasmid construction, see Materials and Methods. PnisA, nisin-inducible promoter; RBS, ribosomal binding site of usp45 gene; SPNuc, SP of staphylococcal Nuc; Nuc, DNA fragment encoding the mature protein. (B) Western blot analysis of L. lactis strains NZ9000(pSEC:Nuc1) and NZ9000(pSEC:Blg41-60:Nuc) using anti-Nuc antibodies. Blg41-60::Nuc production was estimated by Western blot analysis on exponential-phase-induced cultures of lactococcal strains containing pSEC:Nuc1 or pSEC:Blg41-60:Nuc. Protein extracts were prepared 1 h after nisin induction and separated into cellular (C) and supernatant (S) fractions. At an OD600 of 0.5, cultures were induced with 1 ng of nisin per ml of culture. C and S extracts of the induced cultures of NZ9000(pSEC:Nuc1) and NZ9000(pSEC:Blg41-60:Nuc) were hybridized with anti-Nuc antibodies. preNuc, precursor of Nuc; NucB and NucA, mature forms of Nuc; pre41-60:Nuc, precursor of the fusion protein Blg41-60::Nuc; 41-60:Nuc, mature form of Blg41-60::Nuc.