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BTPR_518_sm_suppfig1.doc82KSupporting Figure 1 SDS-PAGE gel of OPH cross-linked to crystallin fibrils (5.8 mg/mL) using pHs 7, 8, and 9 and final concentrations of 2.5, 5, 10 and 20 mM glutaraldehyde (GA). L is the molecular weight standard ladder. Lane 1 contains cross-linked samples at 2.5 mM GA, pH 7. Lane 2 contains cross-linked samples at 5 mM GA, pH 7. Lane 3 contains cross-linked samples at 10 mM GA, pH 7. Lane 4 contains cross-linked samples at 20 mM GA, pH 7. Lane 5 contains cross-linked samples at 2.5 mM GA, pH 8. Lane 6 contains cross-linked samples at 5 mM GA, pH 8. Lane 7 contains cross-linked samples at 10 mM GA, pH 8. Lane 8 contains cross-linked samples at 20 mM GA, pH 8. Lane 9 contains cross-linked samples at 2.5 mM GA, pH 9. Lane 10 contains cross-linked samples at 5 mM GA, pH 9. Lane 11 contains cross-linked samples at 10 mM GA, pH 9. Lane 12 contains cross-linked samples at 20 mM GA, pH 9.
BTPR_518_sm_suppfig2.doc433KSupporting Figure 2 Temperature stability of cross-linked samples using insulin fibrils at 30, 40 and 50 °C, pH 7 and 8. Following the loss of relative activity using the paraoxon assay. Key: (▪) OPH control; (□) OPH-OPH cross-linked using glutaraldehyde; (•) OPH with fibrils and no cross-linker; (ˆ) OPH cross-linked to fibrils with glutaraldehyde. The error bars represent the standard deviation of the mean of three replicates.
BTPR_518_sm_suppfig3.doc128KSupporting Figure 3 SDS-PAGE of the thermostability samples at 30 °C. For each gel the ladder is in kDa. Lane 1 contains OPH at pH 7. Lane 2 contains OPH at pH 8. Lane 3 contains OPH at pH 9. Going from left to right is day 1, 3, 5, 7. Upon investigating the samples from days 0, 3, 5 and 7 using SDS-PAGE (Figure 3) an increase in the band intensity can be seen at ∼42 kDa and ∼35 kDa. These bands correspond to free MBP and free OPH, respectively, suggesting proteolysis of the fusion protein. Free OPH has a higher melting temperature using DSF (Table 3) than the fusion protein which explains the retained activity seen at 30 °C. The dissociation could be due to a protease present in the solution which is selective for the cleavage site located between the OPH and the MBP. The retained activity is not seen at the other temperatures because denaturing of the proteins occurs before the dissociation of the MBP can occur.
BTPR_518_sm_suppfig4.doc189KSupporting Figure 4 Temperature stability of cross-linked samples using crystallin fibrils at 30, 40 and 50 °C, pH 9. Following the loss of relative activity using the paraoxon assay. Key: (▪) OPH control; (□) OPH-OPH cross-linking using glutaraldehyde; (•) OPH with fibrils and no cross-linker; (ˆ) OPH cross-linked to fibrils with glutaraldehyde. The error bars represent the standard deviation of the mean of three replicates.
BTPR_518_sm_supptable1.doc37KSupporting Table 1 Activity of OPH when cross-linked intramolecularly with different concentrations of glutaraldehyde.
BTPR_518_sm_supptable2.doc46KSupporting Table 2 OPH cross-linked to crystallin fibrils (5.8 mg/mL) using pHs 7, 8, and 9 and final concentrations of 2.5, 5, 10 and 20 mM glutaraldehyde. Rates in triplicate were taken initially and after centrifugation.
BTPR_518_sm_supptable3.doc33KSupporting Table 3 Melting temperature of OPH-MBP and OPH from differential scanning fluorimetry. The melting temperature was found by taking the most negative value from the negative differential of the relative florescence units. The error is the standard deviation of the mean. Melting temperature of OPH-MBP and OPH from differential scanning fluorimetry. The melting temperature was found by taking the most negative value from the negative differential of the relative florescence units. The error is the standard deviation of the mean. Melting temperature of OPH-MBP and OPH from differential scanning fluorimetry. The melting temperature was found by taking the most negative value from the negative differential of the relative florescence units. The error is the standard deviation of the mean. Melting temperature of OPH-MBP and OPH from differential scanning fluorimetry. The melting temperature was found by taking the most negative value from the negative differential of the relative florescence units. The error is the standard deviation of the mean.
BTPR_518_sm_supptable4.doc40KSupporting Table 4 Comparison using crystallin and insulin fibrils, of the relative activity of cross-linking samples after the final incubation time of the experiment. O = OPH control, O+G+F = OPH cross-linked to the fibrils using glutaraldehyde.

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