Ni nta agarose resin

A synthetic gene of Top7 was purchased from Eurofins genomics (eurofinsgenomics.jp; accessed on 20 October 2021) and subcloned into a pET28 vector. Expression vectors of the Top7 variants were constructed by a PCR-based mutagenesis using a PrimeSTAR DNA polymerase (Takara Bio Inc., Shiga, Japan). All protein samples were expressed by E. coli BL21(DE3) strain using Studier’s autoinduction medium [13 (link)]. After one-step purification with Ni-NTA agarose resin (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan), the N-terminal hexahistidine tag was removed by thrombin cleavage. The protein samples were re-applied to the Ni-NTA column to remove the cleaved tag and uncleaved protein.
Analytical SEC was performed using a Superdex200 10/300 column (GE Healthcare, Chicago, IL, USA) equilibrated with a 50 mM phosphate pH 7.0, 150 mM NaCl buffer by monitoring 280 nm absorbance.
CD spectra were measured using a CD spectrometer J-820 (JASCO, Tokyo, Japan) with 10 μM protein samples in a 50 mM phosphate pH 7.0, 150 mM NaCl buffer, and 25 °C. A 1 mm quartz cell was used.

To detect PS exposure without extracellular Ca²⁺, a recombinant MFG-E8 D89E fused with 3 × GGGS linker, monomeric EGFP, 3 × GGGS linker, and 8×Histidine, was expressed in CHO cells by the lentiviral system. After infection, highly MFG-E8-expressing cells, corresponding to top 0.5% of GFP-positive cells, were sorted by flow cytometry (FACS AriaII). The sorted cells were seeded into 300 ml of DMEM/Ham’s F-12 (Wako) medium containing 10% FBS (Gibco) and penicillin-streptomycin mixed solution (Nacalai). One day later, the cells reached to 80% confluent, and the medium was changed to a fresh medium containing antibiotics, but not FBS. The cells were cultured for additional four days, with a medium collection performed every two days. The collected medium was mixed with 0.1% TritonX-100 and 5% glycerol, and applied for purification with Ni-NTA agarose resin (Wako). The protein was eluted with 300 mM imidazole, 25 mM Tris (pH 8.0), 150 mM NaCl, and concentrated using Amicon® Ultra (50 kDa cut off) into HBSS buffer. After concentration, 0.1% BSA was added as a carrier and used for the assay of PS exposure.