Purification and characterization of recombinant human fibrinogen.
Our laboratory has been using protein engineering to study the relationship of primary structure to fibrinogen function. In order to examine genetically altered domains in the context of the intact, functional fibrinogen molecule, we have expressed recombinant human fibrinogen in Chinese Hamster Ovary (CHO) cells. The cDNA for each fibrinogen chain was individually cloned into the same expression vector. Each vector was cotransfected with the selection vector pRSVneo into CHO cells. In addition, the plasmids encoding A alpha and gamma were cotransfected with pRSVneo. Cells resistant to G418, a neomycin analogue, were isolated and clonal lines developed. Analysis of these lines demonstrated that CHO cells express and secrete free gamma chain, and an A alpha-gamma complex. To obtain recombinant fibrinogen, the A alpha-gamma G418-resistant clones were transfected with the B beta expression plasmid and a second selection vector, pMSVhis. Colonies resistant to neomycin and histidinol were selected and clonal lines obtained. These clones secreted biologically active recombinant human fibrinogen, which was purified from serum-free culture media by protamine-Sepharose chromatography. Analysis of the purified protein on SDS-polyacrylamide gels demonstrated a pattern indistinguishable from plasma fibrinogen. Removal of Asn-linked carbohydrate with glycosidase F revealed the presence of carbohydrate on the B beta and gamma chains, as is seen for plasma fibrinogen.
Lord
S T
ST
Department of Pathology, University of North Carolina, Chapel Hill 27599-7525.
Binnie
C G
CG
Hettasch
J M
JM
Strickland
E
E
D000818:Animals; D016466:CHO Cells; D002241:Carbohydrates / Q000032:analysis; D006224:Cricetinae; D016895:Culture Media, Serum-Free; D004247:DNA; D005340:Fibrinogen / Q000096:biosynthesis* / Q000737:chemistry; D006801:Humans; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry
D002241:Carbohydrates; D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins; D005340:Fibrinogen; D004247:DNA