A New Functional Model for Prediction of Chaperone Activity of the Recombinant M. tb Acr (α-Crystallin) Using Insulin as Substrate

Figure 2

Expression and purification of acr-pET28a. (a) Expression of acr-pET28a #3 and #6: Lane 1: whole lysate #3; Lane 2: sonicate supernatant #3; Lane 3: sonicate pellet #3; Lane 4: markers 97 kDa, 66 kDa, 43 kDa, 30 kDa, 21 kDa, and 14 kDa; Lane 5: whole lysate #6; Lane 6: sonicate supernatant #6; Lane 7: sonicate pellet #6. (b) Nickel-NTA purification of acr-pET28a: Lane 1: load; Lane 2: flow through; Lane 3: markers 3, 6, 14, 21, 30, and 43 kDa; Lane 4: E1–E3; Lane 5: E5; Lane 6: E6; Lane 7: E7; Lane 8: E8; Lane 9: wash 1 + wash 2 elution gradients E1-E2 (300 mM imidazole), E3 (400 mM imidazole), and E5–E8 (500 mM imidazole). (c) Gel filtration run 2 chromatogram: X axis: UV ₂₈₀ nm; Y axis: elution time (min). (d) Gel filtration Bio-Rad Standards chromatogram: X Axis: UV ₂₈₀ nm; Y Axis: elution time (min); A, aggregates + thyroglobulin 670 kDa, 36.5 ml (73 mins); B, globulin 158 kDa, 44 ml (88 mins); C, ovalbumin 44 kDa, 55 ml (110 mins); D, myoglobin 17 kDa, 77 ml (154 mins); E, vitamin B12 1.5 kDa, 115 ml (230 mins). (e) 15% reducing SDS-PAGE analysis of gel filtration run 1: Lane 1: load; Lane 2: markers 315, 238, 171, 124, 70, 51, 42, 32, 26, and 10 kDa; Lanes 3–10: B4, B6, B7, B8, B9, B10, B11, and B12. (f)15% reducing SDS-PAGE analysis of gel filtration run 2: Lane 1: load; Lanes 2–4: B4, B5, and B6; Lane 5: markers 315, 238, 171, 124, 70, 51, 42, 32, 26, and 10 kDa; Lane 6: B7 and B8; Lane 7: B9; Lane 8: B11; Lane 9: B10. (g) Native-PAGE of his tag elute (H) and gel filtration elute (G): Lanes 1 and 9: BSA control; Lane 3: (H); Lanes 4, 5, and 6: (G); Lanes 7 and 8: (H). (h) Plot of log molecular weight of the different forms of BSA in Native-PAGE. The different sizes of BSA 66, 132, and 198 kDa were plotted against distance migrated and a log mol weight was plotted in MS excel, and the equation was displayed and used to calculate the oligomer size of (H) and (G) samples.

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