Purification and Characterization of AnxA5 and M1234

AnxA5 and M1234 were prepared as described previously[14 (link)]. Briefly, AnxA5 and M1234 were expressed in Escherichia coli M15 (pREP4) (Qiagen, Venlo, the Netherlands), which were transformed with pQE30Xa (Qiagen) containing cDNA of human AnxA5 and M1234. Bacteria were harvested and lysed by sonification. Cell debris was removed by centrifugation. His-tagged proteins were isolated from supernatant by chromatography using nickel columns (GE Healthcare, Eindhoven, the Netherlands) and an imidazole gradient. Purified proteins were checked on homogeneity (MALDI-TOF/TOF) and PS binding activity (ellipsometry) as described elsewhere. All recombinant protein samples contained less than one endotoxin unit per ml as determined by Endosafe PTS spectrophotometer (Charles River, Leiden, the Netherlands).