Recombinant Fusion Antigen Expression in Lactobacillus

To analyze expression of recombinant fusion antigen in L. plantarum, cells from a 50 mL culture were harvested 3 h after induction (25 (link), 43 (link)) and resuspended in 500 μL PBS. Bacterial protein extracts were prepared by disruption in FastPrep tubes containing 1.5 g of glass beads (size ≤ 106 μm; Sigma-Aldrich), using a FastPrep® FP120 Cell Disrupter with a shaking speed of 6.5 m/s for 45 s. After 5 min incubation on ice the shaking process was repeated. The glass beads were removed by sedimentation and the protein extracts were transferred to a new tube. Proteins were separated by SDS-polyacrylamide gel electrophoresis using 10% Mini-Protean TGX Precast gels (BioRad) and transferred to a nitrocellulose membrane using the iBlot^TM Dry Blotting System (Invitrogen). The proteins were detected using the SNAP i.d.® 2.0 Protein Detection System (Merck) using a specific monoclonal mouse anti-ESAT-6 antibody (Abcam) diluted 1:15000 and, subsequently, a polyclonal HRP-conjugated rabbit anti-mouse IgG (DAKO), diluted 1:7500. Proteins were visualized using the SuperSignal™ West Pico Chemiluminescent Substrate (Termo Fisher Scientific) and signals were documented using an Azure c400 system and AzureSpot Analysis Software (Azure Biosystems), following the manufacturer's instructions.