PROCESSING OF HUMAN FACTOR-I IN COS-1 CELLS CO-TRANSFECTED WITH FACTOR-I AND PAIRED BASIC-AMINO-ACID CLEAVING ENZYME (PACE) CDNA

Factor I is an active serine proteinase in plasma that regulates both the classical and alternative complement pathways by cleaving C3b and C4b thereby preventing the assembly of C3 and C5 convertase enzymes. I n this study, a full-length human factor I cDNA was cloned into the pM T2 expression vector and the pMT2-fI construct was expressed transient ly in COS-1 cells and stably in CHO-K1 cells. The transfected COS-1 ce lls secreted large amounts of recombinant pro-factor I (85 kD). Co-tra nsfection of COS-I cells with pMT2-fI and the cDNA expression plasmid for PACE (paired basic amino acid cleaving enzyme), resulted predomina ntly in the secretion of a proteolytically processed form of recombina nt factor I (heavy chain, 47 kD; light chain, 35 kD). Following co-tra nsfection of pMT2-fI and pSVNeo.1 into CHO-K1 cells and selection in m edium containing G418, a stably transfected clone was isolated that se creted pro-factor I (85 kd) and proteolytically processed factor I (he avy chain, 48 kD; light chain, 37 kD) in approximately equal amounts. The molecular sizes of the subunit chains of the expressed factor I we re generally slightly smaller than those of human plasma factor I. The activity of recombinant factor I present in the culture supernatants of transfected COS-I and CHO-K1 cells was assayed by its ability to cl eave I-125-C3b in the presence of factor H and was found to be low whe n compared with factor I purified from human plasma. However, since th e functional activity of purified factor I was reduced approximately 5 0% in the presence of conditioned medium from non-transfected cells, i t is suggested that the cold C3b present in the factor I-deficient ser um used to supplement the culture medium probably competed with the I- 125-C3b tracer, thereby decreasing the sensitivity of the assay for th e recombinant factor I proteins.