This was performed on a gene 9 copy on a pMS119 plasmid using a unique MunI restriction site that was engineered between the codons 2 and 3 to generate gp9MunI. Into this site, DNA fragments encoding the tag sequences were introduced. In addition, longer fragments were introduced which encode two copies of the antigenic tag sequences, resulting in additional 36 and 32 residues in pMS-g9-DT7 and pMS-g9-DHA, respectively. Then, the functionality of the modified proteins was tested by complementation of an M13am9 phage infection (Figure 2 and
3). E. coli K38 bearing the corresponding plasmid was grown overnight in LB medium and plated with top agar containing 1 mM IPTG. After solidification of the top agar 10 μL this website of a phage suspension was applied on top of the agar. Plaque formation was observed after incubation at 37°C overnight. When the cells with pMS-g9-HA were infected with M13am9 clear plaques with a turbid ACY-738 purchase zone were visible on the bacterial lawn (Figure 2). Whereas no plaques appeared with the K38 cells containing the pMS plasmid (Figure 3, panel A), pMS-g7/9 transformed cells showed plaque formation down to the 105-fold
dilution step (panel B). In the absence of IPTG (panel C) plaque formation was observed at the 104-fold dilution which is most likely due to a low expression or to recombination events. When K38 cells with the pMS-g9-T7 (panel D) or with pMS-g9-HA (panel E) were used plaque formation was evident down to the 105-fold dilution step. Similarly, the plasmids encoding the double tags (panels F and G) showed efficient plaque formation, as it was observed on the plates with the suppressor containing E. coli K37 cells (panel H). These results suggest that the gp9 variants expressing the epitope-tagged proteins are functional and allow normal phage propagation. Figure 1 Variants of M13 gp9 proteins. Schematic overview of the gp9 variants used in this work. Into the wild-type a MunI restriction site was introduced between codon 2 and 3 resulting in two additional residues in GPX6 gp9MunI (A). Into this MunI site
short sequences were introduced encoding for the T7 tag in gp9-T7 (B) and for the HA tag in gp9-HA (C). In addition, a double tag was introduced into gp9 generating gp9-DT7 (D) and gp9-DHA (E), respectively. The 4SC-202 cell line protein sequence of each mutant is given in the single letter code. Figure 2 Plaque formation of M13am9 with gp9-HA coat protein. E. coli K38 bearing pMS-g9-HA was mixed with LB top agar containing 1 mM IPTG and poured on an agar plate. After solidification, M13am9 phage was applied and incubated at 37 °C overnight. Figure 3 Complementation of M13am9 infections by plasmid-expressed gp9. E. coli K38 bearing the respective plasmid was mixed with LB top agar containing 1 mM IPTG and poured on an agar plate. After solidification, 10 μL drops of serial diluted M13am9 phage suspensions were applied.