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. In this study, a full-length human factor I cDNA was cloned into the pMT2 expression vector and the pMT2-fI construct was expressed transiently in COS-1 cells and stably in CHO-K1 cells. The transfected COS-1 cells secreted large amounts of recombinant pro-factor I (85 kD). Co-transfection of COS-1 cells with pMT2-fI and the cDNA expression plasmid for PACE (paired basic amino acid cleaving enzyme), resulted predominantly in the secretion of a proteolytically processed form of recombinant factor I (heavy chain, 47 kD; light chain, 35 kD). Following co-transfection of pMT2-fI and pSVNeo.1 into CHO-K1 cells and selection in medium containing G418, a stably transfected clone was isolated that secreted pro-factor I (85 kd) and proteolytically processed factor I (heavy chain, 48 kD; light chain, 37 kD) in approximately equal amounts. The molecular sizes of the subunit chains of the expressed factor I were generally slightly smaller than those of human plasma factor I. The activity of recombinant factor I present in the culture supernatants of transfected COS-1 and CHO-K1 cells was assayed by its ability to cleave 125I-C3b in the presence of factor H and was found to be low when compared with factor I purified from human plasma. However, since the functional activity of purified factor I was reduced approximately 50% in the presence of conditioned medium from non-transfected cells, it is suggested that the cold C3b present in the factor I-deficient serum used to supplement the culture medium probably competed with the 125I-C3b tracer, thereby decreasing the sensitivity of the assay for the recombinant factor I proteins.
Wong
M J
MJ
Department of Cellular and Molecular Pathology, University of Toronto, Canada.
Goldberger
G
G
Isenman
D E
DE
Minta
J O
JO
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D003001:Cloning, Molecular; D017244:Complement Factor I / Q000096:biosynthesis* / Q000235:genetics*; D006224:Cricetinae; D003412:Cricetulus; D045683:Furin; D005822:Genetic Vectors; D000882:Haplorhini; D006801:Humans; D008565:Membrane Proteins; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000096:biosynthesis; D013381:Subtilisins / Q000235:genetics; D014162:Transfection
D008565:Membrane Proteins; D011994:Recombinant Proteins; D013381:Subtilisins; D017244:Complement Factor I; C468731:FURIN protein, human; D045683:Furin