Proteins, protein expression and purification

Endogenous TG (eTG) from human thyroid glands was purchased from Biorad (Hercules, CA). We found it contains some T4, as measured by using our T4-ELISA, but not T3 (Extended Data Figure 6). It is also partially degraded, as shown by SDS-PAGE (Extended Data Figure 2a). While the protein behaved well when performing negative staining EM, it was challenging to produce specimens amenable to high resolution cryo-EM. Therefore, eTG was deglycosylated with the addition of 1 μl/200 μg eTG of PNGaseF from NEB (Ipswich, MA) for 1 h at 37 °C and purified by size exclusion chromatography using a Superose 6 Increase 3.2/300 column (GE Healthcare), equilibrated with buffer T200 (50 mM Tris/HCl, 200 mM NaCl, pH 8.0).

For the recombinant production of human TG (rTG), HEK293T (ATCC no. CRL-1573) cells were cultured as adherent monolayers until 90% confluency in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich Company Ltd., Gillingham, UK) supplemented with 10% foetal calf serum (v/v; Sigma-Aldrich Company Ltd.), L-glutamine and nonessential amino acids (Invitrogen Ltd., Paisley, UK), and transiently transfected with 2 mg of DNA and 4 mg of polyethyleneimine (PEI; Sigma-Aldrich Company Ltd.) per litre of culture. We note that the involvement of glycans in TG dimer formation as revealed by the structure might explain the poor expression yields of thyroglobulin when produced in expression systems with reduced/altered glycosylation, such as insect cells, HEK293S GnT1-/- cells or HEK293T cells in the presence of kifunensine (data not shown).

Five days later, the supernatant containing approximately 0.5 mg/L of secreted TG was harvested and filtered (0.22 μm) for protein purification. For smaller cultures (~125 mL) the supernatant was diluted with an equal volume of buffer T200 containing 20 mM imidazole. For volumes larger than 250 mL the supernatant was concentrated and buffer exchanged into buffer T200 using an Äkta Flux system (GE Healthcare). Subsequently, Ni-NTA agarose beads (QIAGEN, West Sussex, UK) were added to the supernatant (2 mL per litre of supernatant). The mixture was gently stirred at 4 °C for 1 h and the beads collected by centrifugation at 600 xg for 5 min in 50 mL tubes (Falcon, BD Biosciences, Oxford, UK). The beads were poured into a 10 mL EconoColumn (Bio-Rad Laboratories Ltd., Hemel Hempstead, UK) and washed with 10 column volumes (CV) of buffer T200, supplemented with increasing concentrations of imidazole (20 mM, 50 mM, 80 mM), prior to elution with 5 column volumes of buffer T200 supplemented with 500 mM imidazole (all buffers adjusted to pH 8.0). Eluting fractions containing TG, according to SDS-PAGE analysis, were pooled and concentrated prior to further purification by size exclusion chromatography (SEC) using a Superose 6 Increase 3.2/300 column (GE Healthcare), or a Superose 6 10/300 GL column for larger amounts of protein. Fractions containing TG were joined, concentrated by ultrafiltration, aliquoted at a concentration of ~0.5 mg/mL, flash frozen in liquid nitrogen and stored at -80 °C.

For maltose binding protein (MBP) variants, the plasmid was transformed into chemically competent C41(DE3) E. coli cells. 100 mL bacterial cultures were grown in 2xTY medium at 37 °C in the presence of 100 μg/mL ampicillin, and protein expression was induced at an optical density OD₆₀₀ of ~1.0 with 1 mM IPTG for 5 hrs. Cells were harvested by centrifugation at 5000 xg, re-suspended in buffer T200, supplemented with 20 mM imidazole, DNAse, RNaseA, lysozyme (Sigma Aldrich) and protease inhibitors (Roche), and lysed by sonication. The soluble fraction was separated by ultracentrifugation at 40,000 xg and MBP protein variants were purified as described for TG, with a SEC final purification step using a Superdex 200 3.2/300 column.

For human TPO ectodomain expression in insect cells, bacmids were prepared using DH10EMBacY competent cells (Geneva Biotech) following the Bac-to-Bac Baculovirus Expression System user guide by Invitrogen. Sf9 cells were transfected with FuGENE Transfection reagent (Promega). V₁ virus was used for large-scale protein production in 400 mL, and cells were supplemented with 5-aminolevulinic acid, which is a precursor of the heme prostetic group and has been reported to increase specific activity of TPO³⁰. Four days after infection, cells were harvested via centrifugation (800 xg, and then 10.000, both at 4 °C) and the supernatant was concentrated by tangential flow filtration. The concentrated supernatant was dialysed against PBS, pH 7.4 and TPO was purified by nickel affinity purification (GE Healthcare HisTrap HP, 1 mL column) using as binding buffer PBS plus 20 mM imidazole, pH 7.4 and as elution buffer PBS plus 400 mM imidazole, also at pH 7.4. Relevant fractions were pooled, concentrated by ultrafiltration and TPO was further purified using a Superdex 200 10/300 column (GE Healthcare), pre-equilibrated with PBS, pH 7.4. TPO was concentrated to 1 mg/mL and flash frozen in liquid nitrogen for storage at -80 °C. The heme occupancy of TPO was estimated by measuring the ratio of absorbance at 412 nm and 280 nm which was 0.2, which roughly equates to 20% occupancy³⁰.