Bulk production and functional analyses of mouse CD55's native and deglycosylated active domains

We report the use of methylotrophic yeast Pichia pastoris as a host to effi ciently express complement control protein repeats (CCPs) 1-4 of mouse deca y accelerating factor (DAF, CD55) as a soluble protein. With this system, t he 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 afte r 24 h of induction with methanol. A mouse DAF CCP1-4 mutant protein in whi ch its two potential N-glycosylation sites were deleted by changing Asn(187 ) and Asn(262) to Gln was also produced. Using Ni2+-immobilized agarose aff inity chromatography, the recombinant mouse DAF modules with their 6x His t ags could be one-step isolated to SDS-PAGE purity. Polyclonal antibody agai nst native mouse DAF CCP1-4 was raised by immunizing NZW rabbits with the p urified product. Measurements of the bioactivities of the wildtype and muta nt mouse DAF proteins in Cab uptake assays showed no differences in regulat ory activities in either the classical or the alternative pathways. With th e use of the mutant DAF protein, small rod-shaped crystals were produced an d preliminary data obtained. The production of large quantities of function al recombinant mouse DAF CCP1-4 modules and their antibody offers the oppor tunity to study DAF structure and DAF function in vivo. (C) 2001 Academic P ress.