A RAPID METHOD FOR THE ISOLATION OF ANALOGS OF HUMAN CD59 BY PREPARATIVE SDS-PAGE - APPLICATION TO PIG CD59

A method for the rapid isolation of functionally active analogues of h uman CD59 from erythrocytes (E) is described. The method, here applied to pig E, is based on the fractionation of a butanol extract of E gho sts by preparative SDS-PAGE followed by gel filtration on Superose 12. Purification was monitored using a functional complement inhibition a ssay. SDS-PAGE analysis of the product of this procedure indicated a s ingle protein band with apparent M(r) of 20 kDa under reducing and non -reducing conditions. The preparation could be incorporated into guine a pig E to inhibit both CVF-reactive lysis and lysis through C8 and C9 using preformed C5b-7 sites, demonstrating that it contained a CD59-l ike activity. PIPLC treatment of the isolated protein abolished the in hibition. In contrast to SDS-PAGE analysis, amino-terminal sequence an alysis of the preparation showed that it comprised two components. One was identified from databank searches as a fragment of pig glycophori n. These two components could not be separated by either standard or a ffinity chromatographic techniques. The second component was novel and had high sequence homology with human CD59, identifying it as the pig analogue. Further functional studies showed that the pig analogue of human CD59 was effective in the protection of guinea pig E against lys is by serum from a variety of species, including human.