FIG.2.

Zymography of the 3xFLAG fusion proteins. Cell surface proteins from cells expressing the LytF-3xFLAG (A), LytE-3xFLAG (B), and LytC-3xFLAG (C) fusion proteins were extracted as described in Materials and Methods. Equal amounts of proteins (equivalent to 10 OD₆₀₀ units) were applied to the lanes. We confirmed the reproducibility of zymography in at least three independent experiments. (A) Detection of the LytF-3xFLAG fusion protein. Cells were cultured in LB medium at 37°C and were harvested at the transition stage (OD₆₀₀, 2.2). Lane M, size marker; lane 1, wild type; lane 2, E3FL (lytF-3xFLAG); lane 3, WE1 (wprA epr); lane 4, WE1E3FL (wprA epr lytF-3xFLAG). (B) Detection of the LytE-3xFLAG fusion protein. Cells were cultured in LB medium at 37°C and were harvested at the transition stage (OD₆₀₀, 2.0). Lane M, size marker; lane 1, wild type; lane 2, F3FL (lytE-3xFLAG); lane 3, WE1 (wprA epr); lane 4, WE1F3FL (wprA epr lytE-3xFLAG). (C) Detection of the LytC-3xFLAG fusion protein. A lytF mutation was introduced into each strain used for zymography since LytF and LytC overlapped, as described in Materials and Methods. Cells were cultured in LB medium at 37°C and were harvested at the transition stage (OD₆₀₀, 2.4). Lane M, size marker; lane 1, ED (lytF); lane 2, B3FLEd (lytF lytC-3xFLAG); lane 3, WEEd (wprA epr lytF); lane 4, WEB3FLEd (wprA epr lytF lytC-3xFLAG).