Recombinant proteins expression and purification

The DNA fragment encoding PAX8(9–135)-LgBit was PCR‐amplified with primers comprising LguI restriction sites and cloned by Golden Gate into a pET‐derived vector with an N‐terminal His6‐ZZ‐Gly·Ser spacer‐HRV3C affinity purification and solubilizing tag. The DNA fragment encoding PRDM3 (75–434)-SmBit was PCR‐amplified with primers comprising LguI restriction sites for cloning by Golden Gate into a pET‐derived vector with an N‐terminal His6‐StreptagII-spacer‐Rbx‐Gly·Ser spacer‐HRV3C affinity purification and solubilizing tag. The DNA sequence of all expression constructs was verified by Sanger sequencing. The expression plasmids were transformed into BL21 (DE3)-competent Escherichia coli cells (New England Biolabs, Ipswich, MA) and grown overnight at 37 °C. LB medium was inoculated with a bacterial pre‐culture and incubated under constant shaking at 37 °C. At OD600 = 0.8, the culture was chilled to 18 °C, and protein expression was induced by the addition of 1 mM isopropyl β‐d‐1‐thiogalactopyranoside and run overnight. Bacterial cells were harvested by centrifugation at 4000 × g for 20 min, frozen on dry ice, and stored at −80 °C. Recombinant PAX8(9–135)-LgBit and PRDM3(75–434)-SmBit proteins were purified using different protocols.

Cell pellets were thawed and suspended in buffer A (50 mM NaH₂PO₄, 0.3 M NaCl, 30 mM imidazole, pH 7.8) supplemented with cOmplete protease inhibitor (Roche, Switzerland) and TurboNuclease (Merck, Germany). The cells were mechanically disrupted by three passages through an EmulsiFlex C3 homogenizer (Avestin, Canada), and insoluble cell debris was removed by centrifugation for 30 min at 40,000 ×g. The clarified cell lysate was loaded onto 5 ml HisTrap HP columns (GE Healthcare, UK) mounted on an ÄKTA Pure chromatography system (GE Healthcare). Contaminating proteins were washed away with 10 column volumes of buffer A, and the His‐tagged protein was eluted with a linear gradient over 10 column volumes to 100% buffer B (buffer A with 300 mM imidazole). The N‐terminal purification tag was cleaved off overnight at 5 °C by GST-tagged HRV3C protease during dialysis against 20 mM NaH₂PO₄, 150 mM NaCl, 0.5 mM TCEP (tris(2-carboxyethyl)phosphine)), and 10% glycerol, pH 7.5. The dialyzed protein was diluted with an equal volume of 20 mM NaH₂PO₄, 10% glycerol, pH 7.5, before being loaded onto a Mono S 10/100 GL column (GE Healthcare) to remove the cleaved tag, HRV3C protease, and contaminating host cell proteins. The cleaved protein was eluted with a linear gradient of 20 mM NaH₂PO₄, 1 M NaCl, 0.5 mM TCEP, and 10% glycerol, pH 7.5. The fractions containing the PAX8(9–135)-LgBit proteins were pooled, concentrated with Amicon Ultra‐15 10 K centrifugal filter unit (Merck, Germany), and loaded onto a HiLoad Superdex 75 16/600 pg size exclusion column (GE Healthcare, UK) equilibrated with 20 mM HEPES, 150 mM NaCl, 0.5 mM TCEP, and 10% glycerol, pH 7.2. The fractions containing pure protein were pooled and concentrated to ~3 mg/ml in an Amicon filter unit (Merck, Germany). The purity and concentration of the protein samples were determined by reverse-phase ultra-high-performance liquid chromatography (RP-UHPLC), measuring the absorbance at 210 nm. The concentration was calculated using a bovine serum albumin (BSA) standard curve as a reference. Identity and molecular weight of the PAX8(9–135)-LgBit protein was confirmed by LC-MS.

Cell pellets were thawed and suspended in buffer A (50 mM Tris, 300 mM NaCl, 10% glycerol, pH 8) supplemented with cOmplete protease inhibitor (Roche, Switzerland) and TurboNuclease (Merck, Germany). The cells were disrupted by sonication for 5 ×1 min at level 5 and amplitude 50% (Branson sonifier), and insoluble cell debris was removed by centrifugation for 30 min at 40,000 × g. The clarified cell lysate was loaded onto two 1 ml HisTALON columns (GE Healthcare, UK) mounted in series on an ÄKTA Pure chromatography system (GE Healthcare). Contaminating proteins were washed away with 10 column volumes of buffer A, and the His‐tagged protein was eluted with a linear gradient over 10 column volumes to 100% buffer B (buffer A with 200 mM imidazole). The N‐terminal purification tag was cleaved off overnight at 5 °C by His-MBP-3C protease during dialysis against buffer A. The cleaved protein was passed over the re‐equilibrated HisTALON columns to remove the cleaved tag, HRV3C protease, and contaminating host cell proteins. The fractions containing PRDM3(75–434)-SmBit proteins were pooled, and protein concentration was ~0.05 mg/ml. The purity and concentration of the protein samples were determined by RP‐UHPLC, measuring the absorbance at 210 nm. The concentration was calculated using a BSA standard curve as a reference. Identity and molecular weight of the PRDM3 (75–434)-SmBit protein was confirmed by LC-MS.