Escherichia coli bl21 de3 competent cells

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.

Plasmids encoding FOXP3 and 9R-conjugated FOXP3 (FOXP3-9R) were constructed for attachment of 9R to the C-terminus of FOXP3 protein (Supplementary Figure S1a). The plasmids were transfected into BL21 (DE3) competent Escherichia coli cells (New England Biolabs, Ipswich, MA, USA) [13 (link),20 (link)]. Protein expression was stimulated using 0.1 mM isopropyl 1-thio-b-d-galactopyranoside. The expressed proteins were purified using a Ni-nitrilotriacetic acid–agarose column (Invitrogen, Carlsbad, CA, USA), and dialyzed with phosphate-buffered saline (PBS). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed for determining the molecular weight of FOXP3-9R (Supplementary Figure S1b). The concentration of FOXP3 and FOXP3-9R protein was 0.3 mg/mL and 0.2 mg/mL, respectively. We diluted them using PBS to obtain the same concentration (0.1 mg/mL). The proteins were then stored at −80 °C.