FIG. 3.

Expression and subcellular location of a His₆-Sep fusion protein in L. fermentum BR11, L. rhamnosus GG, and L. lactis MG1363. (A) Arrangement of the constructs which were introduced into L. fermentum, L. rhamnosus, or L. lactis. The sep terminator (T sep) and DNA encoding the Sep secretion signal (ssSep), BspA secretion signal (ssBspA), and His₆ (grey box) are indicated. The DNA region which is the site of single-crossover homologous recombination into either the sep or bspA loci of L. fermentum BR11 is spotted and is marked with a cross below. (B) Western blot detection of fusion proteins in cell extracts and in the supernatant using an anti-His₅ antibody. The strains and constructs are indicated at the bottom of each blot. Sizes of molecular mass markers are indicated in kilodaltons on the left. The lanes containing cell extracts prepared by boiling in 2× SDS-loading dye (SDS), by sonication (son), and with 5 M LiCl (LiCl) and the precipitated supernatant fractions (SN) are indicated. The amount of cells or medium loaded in each lane is the equivalent to 500 μl (SDS), 50 μl (son), 160 μl (LiCl), and 675 μl (SN) of culture.