Superdex 200 hr

Recombinant wild type and mutant forms of TTR were expressed and purified from an Escherichia coli cells transformed from the pMMHa plasmid as described previously.^{34 (link)} Briefly, overexpressed proteins in the soluble fraction of the sonicated lysates were extracted using ammonium sulfate precipitation methods. The precipitates were resuspended with deionized water and purified using anion exchange Q column, followed by size exclusion gel-filtration chromatography using Superdex HR 200 column (GE Health care). The expression and purification methods yielded a large quantity of TTR proteins (~ 80 mg from a 1L M9 medium culture). The protein concentration was calculated using an extinction coefficient of 7.76 × 10⁴ M⁻¹ cm⁻¹ at 280 nm.
Amyloid samples were obtained by incubating the protein (0.2 mg/mL) in 200 mM acetate buffer (100 mM KCl, 1 mM EDTA, pH 4.4) for a period of 30 days at 37 °C. The insoluble amyloid was spun down and washed twice with deionized water to remove remaining tetramers and soluble aggregates. The TTR amyloid was examined by transmission electron microscopy (TEM) and thioflavin T (ThT) binding assay in our previous studies.^{33 (link)}