title: Bulk production and functional analyses of mouse CD55's native and deglycosylated active domains.
abstract: We report the use of methylotrophic yeast Pichia pastoris as a host to efficiently express complement control protein repeats (CCPs) 1-4 of mouse decay accelerating factor (DAF, CD55) as a soluble protein. With this system, the mouse DAF CCP1-4-active-domain-containing module linked to a 6x His tag at its C terminus was secreted into the culture supernatant at 15 mg/L after 24 h of induction with methanol. A mouse DAF CCP1-4 mutant protein in which its two potential N-glycosylation sites were deleted by changing Asn(187) and Asn(262) to Gln was also produced. Using Ni(2+)-immobilized agarose affinity chromatography, the recombinant mouse DAF modules with their 6x His tags could be one-step isolated to SDS-PAGE purity. Polyclonal antibody against native mouse DAF CCP1-4 was raised by immunizing NZW rabbits with the purified product. Measurements of the bioactivities of the wild-type and mutant mouse DAF proteins in C3b uptake assays showed no differences in regulatory activities in either the classical or the alternative pathways. With the use of the mutant DAF protein, small rod-shaped crystals were produced and preliminary data obtained. The production of large quantities of functional recombinant mouse DAF CCP1-4 modules and their antibody offers the opportunity to study DAF structure and DAF function in vivo.
authors.affiliation: Institute of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
authors.forename: F
authors.initials: F
authors.lastname: Lin
authors.forename: R M
authors.initials: RM
authors.lastname: Immormino
authors.forename: M
authors.initials: M
authors.lastname: Shoham
authors.forename: M E
authors.initials: ME
authors.lastname: Medof
mesh_terms: D000818:Animals; D000917:Antibody Formation; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018958:CD55 Antigens / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D003179:Complement C3b / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D006031:Glycosylation; D051379:Mice; D009154:Mutation; D010843:Pichia / Q000235:genetics; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D018958:CD55 Antigens; D017931:DNA Primers; D011994:Recombinant Proteins; D003179:Complement C3b