Bl21 star

The DUF283 cDNA, which corresponds to the 128-amino acid (aa) sequence located between 625 and 752 aa of hDicer, was amplified by PCR using a purchased plasmid encoding a complete Homo sapiens Dicer1 ribonuclease type III sequence (PubMed, NM_030621) (GeneCopoeia). The fragment obtained was cloned into the pMCSG7 vector (courtesy of Laboratory of Protein Engineering, Institute of Bioorganic Chemistry, Polish Academy of Sciences), which introduces a His6 tag at the N-terminus of the protein. DUF283 was expressed in E. coli strain BL21Star (Thermo Fisher Scientific) in standard Luria-Bertani (LB) medium. The cells were induced with 0.4 mM IPTG and cultured for 17 hours at 18 °C with shaking. The cell pellets were lysed and purified with Ni²⁺-Sepharose High Performance beads (GE Healthcare) with an imidazole gradient (0.02 M–1 M) in 0.05 mM Tris buffer (pH 8.0) supplemented with 0.5 M NaCl, 0.1% Triton X-100, and 5% glycerol. The next step of purification was performed using a HiTrap Q HP column (GE Healthcare). DUF283 was eluted and then concentrated in a buffer containing 0.05 M Tris (pH 8.0) 0.25 M NaCl, 0.1% and Triton X-100. The protein purity was assessed by SDS-PAGE, and the band corresponding to a putative DUF283 was cut out of the gel (Supplementary Fig. S1) and then analyzed by mass spectrometry (Supplementary Materials).

The chicken FANCM gene was cloned from the chicken DT40 cDNA library. A recombinant plasmid encoding MBP-6×His-TEV protease-recognition site (tev)-FANCM (amino acids 660–804), 6×His-tev-MHF1 (truncated at residue Asn104) and StrepII-tev-MHF2 was used to transform Escherichia coli cells [BL21 Star (Thermo Fisher) with the pRARE2LysS plasmid (Novagen)]. Transformed cells were grown in LB or Terrific Broth containing 1 mM ampicillin at 37°C until the OD₆₀₀ reached 0.7–1.0. Protein expression was induced by the addition of 0.2 mM isopropyl β-d-1-thio­galactopyranoside and the culture was incubated at 20°C for 12–15 h. The cells were harvested by centrifugation using a JLA8.1000A rotor (Beckman) at 4000 rev min⁻¹ for 15 min at 4°C and the bacterial pellet was stored at −80°C until purification.

Plasmid pET15b was used for GTFB [GenBank: AAU08014.2] (C-terminal His-tag) expression in Escherichia coli strain BL21 Star (DE3) (Invitrogen, Taastrup, Denmark).
E. coli strain BL21 Star (DE3) was grown in Luria-Bertani (LB) medium with 100 mg/l ampicillin and 25 mg/l kanamycin medium. GTFB expression was induced using 0.4 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). AVEBE MD20 with DE (dextrose equivalent) value 20 was provided by AVEBE (Veendam, The Netherlands). LiBr was purchased from Fisher Scientific and pullulan standards from PSS (Polymer Standard Service, Mainz, Germany). All other materials and chemicals were purchased from Sigma-Aldrich (St. Louis, US).

The construct of the β-CA sequence from the freeze-dried plasmid supplied by GeneArt was prepared according to the instructions of the manufacturer. The BL21 Star™ (DE3) cells were stored at −80 °C cells (Invitrogen, Carlsbad, CA, USA) and thawed by keeping them on ice. After thawing the competent cells, 25 µl of the cell suspension and 1 µl of the reconstituted plasmid, were transferred into a 1.5 ml centrifuge tube. The suspension was incubated on ice for 30 min. Heat shock was performed by keeping the tube in 42 °C water for 30 s, and transferred immediately on ice for 2 min. 125 µl of SOC Medium (Invitrogen, Carlsbad, CA, USA) was added to the microcentrifuge tube containing the transformed cells, and the tube was incubated at 37 °C for 1 h with shaking (200 rpm). The agar plates containing gentamycin were stored at 37 °C before the transformation. 20 µl or 50 µl of cell suspension described above were spread onto each plate, and the plates were incubated overnight at 37 °C . A volume of 5 ml preculture was prepared by inoculating single colonies from growth plates onto an LB medium with gentamycin (ratio 1:1000), being then incubated overnight at 37 °C with constant shaking of 200 rpm.