Fig. 2

Secretion of LpAsp and LpCht proteins. a Visualization of L. passim expressing GFP, LpAsp-GFP or LpCht-GFP fusion proteins using both visible differential interference contrast (DIC) and fluorescence (Fluorescence) microscopy. Merged images are also presented (Merge). Note that fluorescence signals for LpAsp-GFP and LpCht-GFP were enhanced through longer exposure times compared to GFP alone. Scale bar: 1 μm. b Schematic representation of LpAsp and LpCht proteins. The positions of amino acids encoding SP and molecular functional domain are shown together with those of potential N-glycosylation sites (Y). The total number of AAs in each protein is given at the right. The size of protein is not in scale. c Quantification of intracellular GFP, LpAsp-GFP, and LpCht-GFP via WB analysis of cell lysates, with WT L. passim used as a control. Extracellular protein levels were determined through IP of equal volumes of conditioned medium, followed by WB. Arrowheads indicate two cleaved LpCht-GFP proteins. SDS-PAGE gels stained with InstantBlue displaying the cell lysates and concentrated conditioned medium are presented at the bottom. Equal protein amounts were loaded for the WB or IP analysis. Molecular weights (kDa) of the protein markers are indicated on the left. AA, Amino acid; GFP, green fluorescent protein; IP, immunoprecipitation; LpAsp-GFP, L. passim aspartyl protease–GFP fusion protein; L. passim LpCht-GFP, chitinase-GFP fusion protein; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SP, signal peptide; WB, western blot; WT, wild type