Ni nta

The human BLM (642-1290) (called hBLM-wt for simplicity), and its point mutations of R1000A, R1003A, E1008A, K1009A were expressed and purified as previously described (Guo et al., 2005 (link); Karow et al., 1997 (link)). Briefly, they were cloned into the pET15b-SUMO vector and then expressed in E. coli strain BL21 (DE3). Starter cultures were grown at 37°C and induced with 0.3 mM IPTG at 18 °C for 16 hours. After their SUMO tags were cleaved with Sumo protease, they were purified by fast protein liquid chromatography (FPLC) with sequential chromatography on Ni-NTA (GE Healthcare, Chicago, IL, USA) and cation-exchange chromatography (HiTrap SP, GE Healthcare). The Escherichia coli RecQ helicase was expressed from pET15b-SUMO expression plasmid in Escherichia coli strain BL21 (DE3). After the SUMO tags were cleaved with Sumo protease, then was purified through Ni-NTA column (Teng et al., 2020 (link)). The Gallus gallus WRN (512-1213) helicase was expressed and purified as previously described (Wu et al., 2017 (link)). In brief, it was expressed in E. coli ER2566 cell (NEB) and induced with 0.3 mM IPTG at 18°C overnight. After centrifugation, WRN was purified by FPLC with sequential chromatography on Ni-NTA and Heparin (GE Healthcare). The proteins were stored at −80°C in the storage buffer.

To obtain SUMO-fused peptides for the ITC assay, recombinant proteins were purified first by Ni-NTA (GE Healthcare) and then loaded into a HiLoad 16/600 Superdex 75 column (GE Healthcare) in a buffer containing 200 mM NaCl, 1 mM Ethylenediaminetetraacetic acid (EDTA), and 20 mM Tris-HCl, pH 7.5. The sequences of the SUMO fusion peptides used in the ITC experiments are summarized in SI Appendix, Table S2. To obtain APPBP2-EB-EC for the ITC assay, recombinant proteins were purified first by Ni-NTA (GE Healthcare). TEV protease was added to remove the N-terminal tag, and the proteins were further purified by a HiLoad 16/600 Superdex 200 column (GE Healthcare) in the same buffer containing 200 mM NaCl, 1 mM EDTA, and 20 mM Tris-HCl, pH 7.5. ITC assays were carried out on a PEAQ-ITC calorimeter at 25 °C. ITC experiments were performed by titrating 2 μL of SUMO fusion peptides (0.5 to 1 mM) into cells containing 30 to 50 μM proteins, with a spacing time of 120 s and a reference power of 5 μCal/s. Control experiments were performed by titrating peptides (0.5 to 1 mM) into buffer and were subtracted during analysis. Binding isotherms were plotted, analyzed, and fitted based on a one-site binding model by PEAQ-ITC software (Malvern Panalytical) and Origin Software (MicroCal Inc.).