coli K38 and JS7131. Exponentially growing E. coli K38 cells (panel A) and JS7131 (panel B), respectively, containing the plasmid pMSg9-T7 were pulse-labelled with 35S-methionine for 10 min. The cells were converted to spheroplasts and incubated on ice for 1 h either in the presence or absence of 0.5 mg/mL proteinase K. The samples were immunoprecipitated with antiserum to T7 (lanes 1, 2), to GroEL (lanes 3, 4) and to OmpA (lanes 5, 6), respectively, and analysed on SDS PAGE and phosphorimaging.

(C) The depletion of YidC in the JS7131 cells grown in M9 medium with 0.2% glucose (glc) was verified by Western blot using an antibody to YidC. As control for the non-depleted conditions, the JS7131 cells were grown in the presence of 0.2% arabinose (ara). The insertion of gp9-T7 into the membrane was then investigated in E. coli JS7131. In these cells, the membrane insertase YidC can be depleted when the selleck screening library cells are grown in the presence of glucose [4]. After 2 h growth under glucose conditions the cells were pulse-labelled with 35S-methionine for 10 min and converted to spheroplasts. The protease mapping (Figure 5B) shows Raf inhibitor that the YidC depleted cells did not allow the digestion of the T7-epitope at the N-terminus of gp9 (lane 2). These results suggest that the membrane insertion

of gp9-T7 is YidC-dependent. In both cases, the integrity of the spheroplasts was verified by the protection of GroEL (lane 4) and the proteolytic activity was corroborated by the accessibility of the OmpA protein (lane 6). Assembly of gp9 variant proteins onto phage Assembly of the plasmid-encoded variants onto phage was Flavopiridol (Alvocidib) first followed by dot-blot analysis of phage particles. M13am9 infections in E. coli K38 bearing a plasmid coding for one

of the gp9 variants were performed and the progeny phage were collected and titrated. Equal amounts of phage was applied on nitrocellulose, incubated with antiserum to M13 gp8, to T7 tag or to the HA tag, respectively. The reaction with a secondary peroxidase coupled antibody was analysed by chemoluminescence (Figure 6). Whereas the infecting M13am9 phage reacted only to the anti gp8 serum (panel A), the phage grown in cells with pMS-g9-T7 clearly reacted with the T7 serum (panel B). Similarly, phage from cells expressing the double tag gp9-DT7 also reacted with the serum to the T7 tag. Strong signals were obtained with gp9 proteins with the HA epitopes (panel C) whereas the uninfected K38 cells expressing gp9-T7 or gp9-HA showed only a low signal in the corresponding supernatants. This verifies that the plasmid encoded gp9 proteins with the epitope tags were efficiently assembled onto the phage particles. Figure 6 Presentation of the antigenic tags on gp9 of phage particles. (A) M13 phage (panel A) was applied onto nitrocellulose membrane and incubated with antibody to gp8, T7 tag and HA tag, respectively, at the indicated find more concentrations.