One-step Extraction and Zymographic Analysis of Bacterial Gelatinases

Proteinases of pathogenic bacteria contribute to invasion and pathogenesis by actively degrading the host's extracellular matrix (ECM) components and cellular barriers, modifying the host immune response and degrading the host defense system. This makes the proteinases one of the most virulent molecules and their identification and characterization are essential for preventing and controlling disease. Many bacteria also possess outer membrane-bound proteinases. Isolation and characterization of outer membrane proteins in their active forms present significant challenges due to their localization within the outer membrane. In this study, we demonstrate a single-step method for extracting both cytoplasmic and outer membrane-associated gelatinases from Leptospira and demonstrate their enzymatic activity using zymography. In this method, we use sodium deoxycholate to disrupt the cell membrane and release the bound proteins and 6-aminocaproic acid to solubilize the released membrane protein in its native form. Further, we show a zymographic analysis of the leptospiral gelatinases. This approach provides a versatile framework for isolating, purifying, and characterizing metalloproteases, collagenases, and gelatinases from various bacterial pathogens.