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As for NupC-V532C, samples of detergent solubilised AcrB were aspecifically adsorbed onto the surface of a Nunc Maxisorp® plate and probed with DARPins. Two of these DARPins (A1 and A2) were selected against AcrB SMALPs and the ELISAs (see Figure 5.11) show that despite the lack of commonality in the sequences of these two DARPins (see Figure 5.7) both produced similar binding curves. However neither DARPin saturated binding at 10 µM but both have similar adsorption values at this concentration. As for MPSIL0294-V532C, negative controls were measured without target protein and the data sets were analysed for a significant difference using a two tailed T-test. Despite the similarities between the two traces. In the A2 trace the data points might start to level off typical of an IC50 plot. However, the low

absorbance values and standard deviations in the data points, makes an unambiguous assessment impossible and for both DARPins only a lower limit can be determined for the IC50

of 100 nM.

Figure 5.11 DARPin binding ELISA performed against DARPins selected against AcrB SMALPs: 10 µg of detergent solubilised AcrB was aspecifically adsorbed onto the surface of Nunc Maxisorp® plates and treated with a serial dilution of DARPin A1 and A2 from 10 µM – 1 pM. The DARPins showed an ability to bind AcrB SMALPs particularly tightly after two rounds of phage display and two rounds of subsequent validation. The plates were probed with streptavidin-HRP and the absorbance at 450 nm was analysed after incubation with TMB and subsequent exposure to 0.5 M H2SO4.The negative control for both ELISAs was the DARPin without the target

protein present and all of the readings have been adjusted for background signals by subtracting the absorbance reading produced by BSA alone. Each ELISA was also repeated three times with duplicates of each concentration, error was calculated by the standard deviation of all six of these data points and the significance between the test data and negative control was calculated by a 2 tailed t-test. Data points which are significantly different from the negative have been highlighted with a green asterisk.

The traces produced by ELISAs performed on the DARPins selected against AcrB nanodiscs (see

Figure 5.12) show that all of the DARPins are able to bind to detergent solubilised AcrB to

some extent, however, the best binder when both statistical significance and A450nm readings

are taken into account is E3. When compared in terms of significance, D1 is the best as it is significantly different from the negative control at every concentration other than 100 nM, F1 is only significantly different from the negative control down to a concentration of 100 nM, H1s significance ceases after 1 µM, and finally E3 is significantly different from its negative control down to 10 nM. The highest absorbance reading however, shows that E3 performs the best out of the DARPins. When 10 µM of DARPins is used on the ELISA the reading produced by E3 is substantially higher (approximately twice as large) than D1, F1 and H1. Like all of the other ELISAs performed in this Chapter, negative controls were performed without membrane protein present. The negative controls of H1 and F1, however, suffers from a higher than usual degree of fluctuations. This effect is aggravated in the graph due to the small values of the absorbance readings in these results.

Figure 5.12: DARPin binding ELISA performed against DARPins selected against AcrB nanodiscs: This ELISA was carried out on 10 µg of immobilised AcrB solubilised with DDM using a serial dilution of DARPin from 10 µM to 1 pM. Each ELISA was repeated three times and each concentration was tested in duplicate, the negative control was the DARPin tested in the absence of AcrB and background noise was corrected for by subtracting all of absorbance readings from those produced by BSA alone. Statistical significance was determined by two tailed t- test, all data points which are significantly different from their corresponding negative control are highlighted with a green asterisk.

5.5.3 DARPins selected against NupC SMALPs

The traces produced by the DARPins selected against NupC SMALPs show that 5 of the 6 DARPins successfully bind to the target. DARPin A3 fails to follow the expected data trend but remains significantly different from the negative control throughout the serial dilution, suggesting a concentration independent binding although considering the A450nm readings it is

likely that this arbitrary binding is quite poor. All of the DARPins show a significant increase over the various negative controls at most concentrations, more so than the other DARPins which have been tested in this manner. The exception being H10, the negative control of which comes into closer proximity with the test data than any of the other NupC DARPins, thus suggesting that the DARPin does not bind to NupC particularly well. As with the other ELISAs, the data fails to plateau and therefore an affinity for the DARPins cannot be determined. Instead a lower estimate of 100 nM for DARPins C2, G2, H8 and H10 can be given. The traces produced by DARPins A3 and A5 however do not have allow a clear estimate to be given.

When the data trends along with the absorbance readings are considered, C2 and G2 appear to be the most promising DARPins and could be used in co-crystallisation trails (see Figure

5.13).

Figure 5.13: DARPin binding ELISA performed against DARPins selected against NupC SMALPs: This ELISA was carried out on 10 µg of immobilised NupC solubilised with DDM using a serial dilution of DARPin from 10 µM to 1 pM. Each ELISA was repeated three times and each concentration was tested in duplicate. The data has been compared with a negative control which consisted of the DARPin tested in the absence of NupC and background noise was corrected for by subtracting all of the absorbance readings from those produced by BSA alone. Statistical significance was determined by two tailed t-test, all data points which are significantly different from their corresponding negative control are highlighted with a green asterisk.

5.5.4 DARPins selected against VcCNT SMALPs

Samples of detergent solubilised VcCNT were produced via the protocol described in section

2.5.3, as shown in Figure 5.14 samples taken throughout the purification procedure were

analysed via SDS-PAGE and western blotting. As was the case with previously described purifications, the samples taken throughout the procedure give an insight into the state of the protein. From Figure 5.14 it is evident that VcCNT expresses well (see intense band in the ‘Total’ sample of the Coomassie stained gel and its counterpart on the western blot). The vast majority of this protein is successfully solubilised and only a minimal amount of VcCNT did not successfully bind to the resin. The final yield of VcCNT was determined to be 4.35 mg from 100mg of membrane produced in a 30 L fermentation after extensive dialysis and

concentration in a viva spin concentrator with a 60 kDa molecular weight cut off. Like the other ELISAs, the absorbance readings produced at 450 nm have been plotted and compared with a negative control consisting of the DARPin analysed in the absence of

immobilised target protein (Figure 5.15). All five DARPins produce statistically significant data above 100 nM except for C1 which is only significantly different from its negative control at 10 µM. When the error of H11 is considered it appears to be the most consistent DARPin out of those chosen for further study (along with E3, C2 and G2 in Figure 5.12 and Figure 5.13 respectively), as the level of error is minimal across the entire data set. H11 also produces the highest absorbance reading with 10 µM of DARPin out of all 20 DARPins studied, with a value of 0.345. Unfortunately like the previously discussed ELISAs, the range of concentrations used in Figure 5.15 is not wide enough to produce a plateau in the trace therefore an affinity cannot be determined this way and for all five DARPins only a lower value of 1 M can be given.

Figure 5.14: IMAC Purification of detergent solubilised VcCNT: Samples taken throughout the purification of VcCNT were analysed via 12% SDS-PAGE stained with Coomassie blue and western blotting probed with an anti- polyhistidine tag antibody conjugated with HRP. Each sample was normalised to a volume of 10 µL, therefore the ‘Total’ ‘Supernatant’ and ‘Flow Through’ represents 0.02% of the original sample. The ‘Pellet’ represents 0.1% and the two washes represent 0.05%. The eluted protein was subjected to extensive dialysis and concentration in a viva spin concentrator with a 60 kDa molecular weight cut off, it represents 0.33% of the original sample.

Figure 5.15: DARPin binding ELISA performed against DARPins selected against VcCNT SMALPs: The ELISAs were performed in the presence of 10 µg of detergent solubilised VcCNT which was aspecifically adsorbed onto the surface of a Nunc Maxisorp® plate and probed with a serial dilution of each DARPin ranging from 10 µM to 1 pM. The negative control consisted of each DARPin analysed in the absence of target protein and all data points were subtracted from the background noise, determined by the analysis of BSA in the absence of DARPin. Each ELISA was repeated three times and each concentration was tested in duplicate in order to calculate the error by standard deviation. Statistical significance was determined by a two- tailed T-test and all data which is statistically significant from its negative control counterpart is highlighted with a green asterisk.