K38 cells expressing the wild-type gp9 from the plasmid (B) showed plaque formation at the 105-fold dilution, similar to the suppressor cells K37 (H). When no IPTG was added to the plate plaque formation was reduced (C). Cells expressing the modified gp9 proteins all showed efficient plaque formation. Gp9-T7 (D), gp9-HA (E), gp9-DT7 (F) and gp9-DHA (G) were analysed. Expression of the modified gp9 protein in E. coli The plasmid-encoded gp9 variants were analysed for expression in E. coli K38. The cells were grown

at 37°C to the early exponential phase in M9 minimal medium. Protein expression was induced by adding 1 mM IPTG and after 10 min the newly synthesised proteins were pulse-labelled for 10 min with 35S-methionine. The total bacterial MLN8237 price proteins were TCA precipitated to remove the non-incorporated 35S-methionine and immunoprecipitated using an antiserum to the T7 tag or to the HA tag, respectively (Figure 4). Since gp9 is a very small protein of 32 amino acids containing only two methionines the protein band on a SDS tricine PAGE is difficult to visualise. When comparing the protein pattern of cells expressing gp9-T7 (lane LY2874455 3) with cells containing

the empty plasmid (lane 2) a protein band of about 5.5 kDa was observed. Also a weak band of gp9-HA (lane 4) was visible on the gel. The size of the protein was estimated in relation of the major coat protein gp8 shown in lane 1. Since the 50 amino acid residues long gp8 has a molecular weight of 5.2 kDa, the gp9-T7 with 51 residues and gp9-HA with 49 residues are proteins of very similar molecular weight. Figure 4 Expression of gp9-T7 from a plasmid. Exponentially growing E. coli K38 cells bearing a plasmid encoding M13 gp8 (lane 1), the empty pMS plasmid (lane 2), pMS-g9-T7 (lane 3) and pMS-g9-HA (lane 4), respectively, were induced for 10 min with IPTG and pulse-labelled with 35S-methionine for 10

min. The proteins were precipitated with trichloroacetic acid (TCA) and immunoprecipitated with antiserum to Methamphetamine gp8 (lane 1), to T7 (lane 2, 3) and to HA (lane 4), respectively. SDS tricine PAGE was used to separate the proteins and the radioactivity was visualised by phosphorimaging. Membrane insertion of gp9-T7 The membrane insertion of gp9 with the N-terminal T7 tag was analysed in E. coli K38 cells bearing the pMS-g9-T7 plasmid. The gp9-T7 protein was expressed as described above. The cells were converted to spheroplasts and analysed by protease mapping (Figure 5A). The protein immunoprecipitated with antiserum to the T7 tag was readily digested by proteinase K added to the outside of the spheroplasts (lane 2). This suggests that the Eltanexor manufacturer antigenic tag of gp9 was accessible to the protease at the periplasmic surface, whereas the cytoplasmic GroEL protein was protected from digestion (lane 4). Further, the periplasmic portion of the OmpA protein was digested by the proteinase K (lane 6) confirming the proteolytic activity.