Production and functional characterization of a soluble recombinant form of mouse CD59.
This report describes the engineering, expression, purification and functional characterization of a soluble recombinant form of murine CD59 (srMoCD59). We report the expression in Chinese hamster ovary (CHO) cells of a modified mouse CD59 cDNA that had been truncated at D-74, resulting in the loss of the glycosylphosphatidyl inositol (GPI) anchor, and containing six additional C-terminal histidines. The expressed srMoCD59 was purified from tissue culture supernatant by means of its poly-histidine tag using immobilized metal affinity chromatography. In comparison with CD59 on mouse erythrocytes, the srMoCD59 had a reduced molecular weight (18-20 000 as compared with 20-28 000 for GPI-anchored srMoCD59). The terminal complement inhibitory capacity of this soluble recombinant protein was assessed using two methods: a cobra venom factor (CVF)-triggered 'reactive-lysis' system and a C5b-7 site assay. In both assays, srMoCD59 inhibited lysis by the sera from all three species tested in the rank order mouse > rat >> human. The amount of srMoCD59 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 presence of serum markedly interfered with the activity of srMoCD59 and have important implications for the use of recombinant soluble CD59 analogues as therapeutic agents in complement-mediated diseases.
Rushmere
N K
NK
Department of Medical Biochemistry, 3rd Floor Tenovus Building, University of Wales College of Medicine, Heath Park, Cardiff, UK.
Van Den Berg
C W
CW
Morgan
B P
BP
D000818:Animals; D018957:CD59 Antigens / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003169:Complement Inactivator Proteins / Q000276:immunology; D006224:Cricetinae; D003412:Cricetulus; D006461:Hemolysis / Q000276:immunology; D006801:Humans; D051379:Mice; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D020539:Sequence Analysis, Protein; D012995:Solubility; D013045:Species Specificity
D018957:CD59 Antigens; D003169:Complement Inactivator Proteins; D011994:Recombinant Proteins