Production and functional characterization of a soluble recombinant form ofmouse CD59

This report describes the engineering, expression, purification and functio nal characterization of a soluble recombinant form of murine CD59 (srMoCD59 ). We report the expression in Chinese hamster ovary (CHO) cells of a modif ied mouse CD59 cDNA that had been truncated at D-74, resulting in the loss of the glycosylphosphatidyl inositol (GPI) anchor, and containing six addit ional C-terminal histidines. The expressed srMoCD59 was purified from tissu e culture supernatant by means of its poly-histidine tag using immobilized metal affinity chromatography. In comparison with CD59 on mouse erythrocyte s, the srMoCD59 had a reduced molecular weight (18-20 000 as compared with 20-28 000 for GPI-anchored srMoCD59). The terminal complement inhibitory ca pacity of this soluble recombinant protein was assessed using two methods: a cobra venom factor (CVF)-triggered 'reactive-lysis' system and a C5b-7 si te assay. In both assays, srMoCD59 inhibited lysis by the sera from all thr ee species tested in the rank order mouse > rat >> human. The amount of srM oCD59 required to produce 50% inhibition of lysis in the C5b-7 site assay, using purified terminal components to develop lysis, was 10-fold less than that required in the same assay when EDTA serum was used as a source of C8 and C9, or in the CVF reactive lysis system. These data indicate that the p resence of serum markedly interfered with the activity of srMoCD59 and have important implications for the use of recombinant soluble CD59 analogues a s therapeutic agents in complement-mediated diseases.