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From the data described above it was apparent that the phage clones isolated bound NS7 with varying degrees of affinity. Next, we wanted to confirm the binding specificity of all the phage clones isolated against NS7 as our final aim was to identify a peptide with high specificity to MNV NS7. Therefore, a cross binding assay measuring the ability of each phage clone to bind to a variety of antigens was performed by ELISA. In the cross binding assay, 5 X 10¹¹ pfu/ml phage clones were exposed to different targets, which were immobilised on the ELISA plate (Figure 4.7).

From this assay, a more complex binding pattern was observed. All the NS7 phage clones have an affinity not only for the NS7-his used during their initial selection, but also to the untagged Poliovirus 3D RNA polymerase (PV3D), untagged Lordsdale virus VPg (LDV VPg), his-tagged Feline calicivirus protease-polymerase (FCV p76) and Cherry tagged MNV VPg-his (VPg). However, they clearly lack the ability to bind streptavidin, BSA (a non-recombinant protein) and 4E-BP1 proteins (Figure 4.7). As a positive control, the streptavidin-binding phage clone (SA) bound specifically with high affinity to streptavidin. A binding pattern to other recombinant proteins similar to that observed for the phage selected against NS7 was also observed for streptavidin-binding phage clone (Figure 4.7). This non-specific streptavidin-binding to the recombinant proteins is presumably due to the presence of a certain ‘factor’ that is not present in the non-recombinant protein i.e. Streptavidin or BSA.

All the proteins used in the cross binding assay above were recombinant proteins previously purified in our lab, except streptavidin and BSA. Since the recombinant proteins were expressed and purified from E. coli, there could also be contaminants from bacterial proteins that might be co-purified along with the target protein. This would then explain the non-specific binding of the isolated phage to other recombinant proteins in the cross binding assay. If this was the case, then the co-purifying bacterial proteins may be present in some recombinant proteins used in this assay. To assess this, all the recombinant proteins (used in the cross binding assay) were resolved on a gradient 4–12% PAGE gel and subjected to colloidal commassie blue staining, a very sensitive staining method, to visualise the protein bands (Figure 4.8). This sensitive method of staining was used to carefully analyse whether there were any co-purified contaminant proteins from bacterial cells in each purified proteins. Figure 4.8 indicated that there were no obvious commonly co-purified contaminating proteins from E. coli present in the recombinant proteins tested. However, low levels of other contaminants may have been present. The small

Figure 4.7 Cross binding assay of phage clones against different targets. ELISA was carried out by immobilising 0.4 µg of MNV NS7-his (NS7), untagged Polio 3D (PV3D), untagged Lordsdale VPg (LDV VPg), FCV p76-his (FCV p76), 4E-BP1-his (4E-BP1), Cherry MNV VPg-his (VPg), streptavidin (Strep) and BSA across the ELISA plate. 5 x 10¹¹ pfu/ml of purified single clone phages were subjected to the binding reaction with respective targets in the ELISA plate. After extensive washes with TBST, the binding affinity was detected using HRP-conjugated anti-M13 antibody. The TMB solution was used as the substrate, the reaction was stopped by adding HCl and the plate was then read at 450 nm. The streptavidin-binding peptide phage clone (SA) was used as the positive control. The reactions were carried out in triplicate and the error bars represent standard deviation.

multiple bands observed in Figure 4.8 may represent breakdown or degraded products from the purified recombinant protein. It is possible that we may have co-purified some unidentified ‘factors’ other than protein that the phage could bind to.

Examples include endotoxin (a complex of lipopolysaccharides [LPS]) (Cardoso et al., 2007; Feng et al., 2007) or nucleic acid, both of which cannot be detected on the SDS PAGE gel. This could possibly explain why all the isolated clones bound to the recombinant proteins but not to streptavidin or BSA. Moreover, the streptavidin-binding clone (SA), despite having specific streptavidin streptavidin-binding actually also bound to the various recombinant proteins with the same relative binding pattern as seen for the phage specifically isolated against NS7 (Figure 4.7). This suggests that the isolated phage bound non-specifically to other factors that may have been co-purified during the bacterial protein preparation.

We concluded that the phage clones isolated by panning against NS7-his bind non-specifically to NS7. This may partially be due to the general elution buffer used during the panning procedure, which resulted in the elution of the non-specific binders from the binding reaction. As a result, it was hypothesised that using a more specific elution during the panning process was likely to represent the best method of obtaining specific binding phage. Therefore, we decided to use a more robust purification procedure to purify untagged forms of recombinant NS7 and VPg then use known ligands of NS7 (i.e.; VPg, NS7 antiserum and viral RNA) as a form of

Figure 4.8 Recombinant proteins used in the cross binding assay. SDS PAGE analysis of recombinant proteins that were used in the cross binding analysis of the identified peptide phage. 5µg of each protein was resolved on a 4 – 12% reducing gradient PAGE gel and subjected to colloidal coomassie blue staining. M-marker, NS7-MNV NS7-his (58.4kD), PV3D-untagged tagged Polio 3D (50.7kD), LVPg-untagged LDV VPg (15kD), p76-FCV p76-his (76kD), MVPg-Cherry MNV VPg-his (26kD), 4E-4E-BP1-his.

specific elution during the phage display panning procedure. The purification of untagged versions of recombinant NS7 was performed since the use of fusion proteins (e.g. GST fusions) as a target in phage display selection has been shown to potentially generate false positive results (Murthy et al., 1999).

4.2.2 Expression and purification of untagged recombinant MNV NS7