Fig 1.
Workflow of casp-6 sample preparation and optimization for NMR spectroscopy.
Each step in the protein production, purification, and exchange process was explored and assessed by multiple tests. Key areas of manipulation are noted.
Fig 2.
Determination of ideal E. coli growth conditions for casp-6 overexpression in minimal media.
(A) Bacterial cell culture process comparing growth of BL21 (DE3) E. coli in M9 (M9) or 2x M9 minimal media (2x M9) to 2xYT rich media. (B) Protein yield was dependent on culture media used (2x M9: n = 8, 2xYT: n = 8, M9: n = 3 independent purifications). Casp-6 yields were calculated following a standardized purification protocol. (C) Growth rates of BL21 (DE3) E. coli were similar in M9 (triangles) and 2x M9 (circles) minimal media in three independent experiments. (D) Protein yield following purification was measured as a function of induction OD600. (E) Bacterial pellet mass was measured as a function of induction OD600.
Fig 3.
Comparison of anion-exchange purification protocols to balance elution concentration and purity.
(A) Overlay of four anion-exchange chromatograms on a 5mL HiTrap Q column (Cytiva Life Sciences) tracked by NaCl concentration dashed lines, left Y axis represents percent of the total protein obtained for each purification scheme tested. (B) Comparison of four anion-exchange protocols tested illustrates benefits of lowered flow rate and step elution profile in both % Total Protein and NaCl concentration in the elution profile (black line). Fraction numbers excluded for clarity. (C) Purity of casp-6 following anion-exchange was assessed by SDS-PAGE and did not significantly improve from that obtained from Ni-affinity chromatography on a 5 mL HisTrap HP column (Cytiva Life Sciences) when followed by anion-exchange on a 5 mL HiTrap HP Q Anion-Exchange column (Cytiva Life Sciences). Fractions from the purification include cell debris following cell lysis and centrifugation (CD), cell lysate (Lys), Ni column flow-through (NiFT), Ni column wash (Ni Wash), Ni column elution (Ni Elu), anion-exchange flow-through (QFT), anion-exchange wash (Q Wash), as well as individual fractions from anion-exchange. (D) Purity of casp-6 following solely Ni-affinity purification was assessed by SDS-PAGE and found sufficient for NMR following buffer exchange. Fractions from the purification include cell debris following cell lysis and centrifugation (CD), cell lysate (Lys), Ni column flow-through (NiFT), Ni column wash (Ni Wash), as well as individual fractions from the Ni-affinity elution. (E) PD-10-desalting-column buffer exchange using a high initial concentration of casp-6 yielded a higher final concentration and improved recovery. Two different samples were analyzed. One pool included half of fraction A5 and A8-A10 from the Ni-affinity purification shown in (D). The second pool included half of A5 and A6-A7 from the Ni-affinity purification shown in (D). Individual fractions from the PD-10 desalting column elution are also shown.
Fig 4.
Effects of buffer components and desalting column size on percent recovery following buffer exchange.
(A) Casp-6 recovery following buffer exchange on NAP-5 or PD-10 Sephadex-G25 desalting columns (Cytiva Life Sciences) under conditions listed was estimated by spectroscopic absorption at 280 nm. (B) Cumulative differences between various exchange conditions summarizing the results of the 17 individual exchange protocols tested in (A) assessed buffer and pH, presence of DTT, glycerol or salts and desalting column sizes (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Bars represent mean ± standard deviation (n = 3-9). Bars are colored by condition category. Together these suggested superior casp-6 recovery in Tris pH 8.5, with DTT and glycerol, using a PD-10 column.
Fig 5.
Impact of buffering agents and additive on casp-6 activity.
(A) Cleavage of the fluorogenic casp-6 substrate VEID-amc was monitored in the presence of various buffer additives and buffering agents. Activity was greater in HEPES pH 7.5 and phosphate pH 7.4 than in Tris pH 7.5. (B) Buffer additives and buffering agents showed no statistically significant differences between different buffer additives. (C) Statistically significant differences in casp-6 activity were observed between buffering agents and NaCl concentrations within sodium acetate pH 4.6 and Tris pH 7.5 buffer conditions. (D) Statistically significant differences were not observed between NaCl concentrations when combining results from various buffering agents. Bars represent mean ± standard deviation. All conditions were tested in two technical replicates and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 6.
1H NMR spectral quality as a function of varying buffer conditions.
(A) Impact of buffering agents Tris pH 7.5, phosphate pH 7.4, and HEPES pH 7.5 was assessed by monitoring amide and methyl peak dispersion in the casp-6 D179 CT 1H NMR spectrum measured at 600 MHz. (B) Increasing NaCl concentration resulted in <5% variation in peak height or linewidth in the casp-6 D179 CT 1H NMR spectrum. (C) Presence of octyl-β-glucoside (BOG) did not significantly improve casp-6 D179 CT 1H NMR peak resolution. (D) Casp-6 FL C163S showed superior dispersion of amide peaks while the spectrum of casp-6 D179 CT showed greater methyl peak dispersion. The presence of glycerol did not produce notable spectral improvement for either casp-6 construct.
Fig 7.
Optimized NMR sample preparation protocol for casp-6 D179 CT.
The optimal preparation protocol for casp-6 NMR consisted of growing a large culture of casp-6 D179 CT in 2x M9 to an OD600 of 1.1 prior to induction, purification by Ni-affinity chromatography, and buffer exchange via a PD-10 desalting column into a buffer containing 20 mM d-Tris pH 8.5, 200 mM NaCl, 5% d-glycerol, 10 mM d-DTT, and 100% D2O. NMR assignments for the 13C-Ileδ1-methyl labeled casp-6 D179 CT 1H-13C HMQC spectrum can now be found at BMRB entry 53341 in the biological magnetic resonance bank.