Bound proteins were incubated in 50 mM Tris–HCl buffer (pH 7.5) containing 300 mM NaCl (buffer A) with thrombin (10 U/mg, GE Healthcare) at 4°C for 12 h to cleave the hexa-histidine and gluthathione S-transferase moieties, respectively. Released proteins were dialyzed in buffer B (50 mM Tris–HCl [pH 8.0] containing 150 mM NaCl and 1 mM DTT) and stored at 4°C for use
within the next 48 hours. A 100-μl volume of each recombinant protein (~100 μg) was loaded onto a SuperdexTM 75 10/300 GL (GE Healthcare) in buffer B at 4°C. The chromatography #4SC-202 randurls[1|1|,|CHEM1|]# was performed at a flow rate of 0.5 ml/min, and fractions of 0.5 ml were collected and analyzed by SDS-PAGE. The gel filtration column was calibrated by running a set of protein standards (Aldolase, 158 kDa; Conalbumin, 75 kDa; Ovalbumin, 43 kDa and Myoglobin, 17 kDa). Rabbit polyclonal antibodies raised against full-length EssB were purified
prior to use in immunoblot experiments as described earlier [20]. Transmission electron microscopy (TEM) and image processing Purified recombinant proteins EssB and EssBΔM were prepared as described above, dialyzed in Buffer B (without DTT) and diluted to approximately 10 to 50 μg/ml. Proteins were APR-246 research buy bound to glow discharged, carbon coated (Edwards Auto 306 Evaporator) copper grids (400 mesh), washed, and subsequently negatively stained using 2% uranyl acetate (Electron Microscopy Services). Images were recorded using a Tecnai F30 (Philips/FEI) transmission electron microscope (Field emission gun, 300-kV accelerating voltage, with a magnification of 49,000 to 75,000×) and a high performance CCD camera with a 4k × 4k resolution.
Images ID-8 were captured using Gatan DigitalMicrograph software and processed using Adobe Photoshop (Adobe, San Jose, CA, USA). Images of single protein were selected manually. Acknowledgements and funding The authors thank Olaf Schneewind for careful reading of the manuscript, Khaled Aly and members of the Schneewind and Missiakas laboratory for suggestions and discussions. The authors are grateful for comments provided by the referees and help with BLAST analyses. Mark Anderson acknowledges support by the Biodefense Training Grant in Host-Pathogen Interactions T32 AI065382 at the University of Chicago and American Heart Association award 11PRE7600117. This work was supported by the National Institute of Allergy and Infectious Diseases, Infectious Diseases Branch (award AI 75258) to DM. References 1. Dalbey RE, Wickner W: Leader peptidase catalyzes the release of exported proteins from the outer surface of the Escherichia coli plasma membrane. J Biol Chem 1985, 260:15925–15931.PubMed 2. Emr SD, Hanley-Way S, Silhavy TJ: Suppressor mutations that restore export of a protein with a defective signal sequence. Cell 1981, 23:79–88.PubMedCrossRef 3. Oliver DB, Beckwith J: E.