Abstract
Expression vectors containing full-length cDNAs for each of the human fibrinogen chains were constructed. COS-1 cells were transfected with single vectors, mixtures of two, or with all three vectors and stable cell lines selected. Cells transfected with single vectors, or with mixtures of any two vectors, expressed the appropriate fibrinogen chains but did not secrete them. COS cells transfected with three vectors expressed all of the chains and secreted fibrinogen. COS cells transfected with three vectors contained, intracellularly, a mixture of fibrinogen-related proteins. The four main intracellular products were nascent fibrinogen, an A alpha.gamma complex, free A alpha chains, and free gamma chains. This is a similar pattern to that noted in Hep G2 cells. The intracellular forms of fibrinogen were sensitive to endoglycosidase H, indicating that they reside in a pre-Golgi compartment. Secreted fibrinogen was endoglycosidase H-insensitive, suggesting that the secreted glycoprotein moieties were processed in the normal manner. When mixed with plasma fibrinogen, radiolabeled recombinant fibrinogen was incorporated into a thrombin-induced clot. These studies demonstrate that COS cells transfected with all three fibrinogen chain cDNAs are capable of assembling and secreting a functional fibrinogen molecule.
Highlights
Our studies are aimed at determining how this multichain protein is synthesized, assembled, and secreted
The To determine whether the Aor and y cDNAs were expressed in correct orientation was determined by digesting the plasmid DNA COS-1 cells, the cells were first transiently transfected with either with BamHI/HindIII and selecting the clones that yielded fragments pBC12BI-Aa or pBC12BI-y by the calcium phosphate method [21]
Expression of Single and Combinations of Fibrinogen Chain cDNAs by COS Cells-COS-1 cells were transfected with the expression vector pBC12BI containing either full length ACY
Summary
Blood Center, 310 E. 67th St., New York, NY 10021. Tel.: 212-5703059: Fax: 212-570-3195. The To determine whether the Aor and y cDNAs were expressed in correct orientation was determined by digesting the plasmid DNA COS-1 cells, the cells were first transiently transfected with either with BamHI/HindIII and selecting the clones that yielded fragments pBC12BI-Aa or pBC12BI-y by the calcium phosphate method [21]. As described above the linear vector containing y cDNA was processed to form the circular expression vector (Fig. ID).The correct orienmethionine and the expressed fibrinogen chains determined by immunoprecipitation, SDS-polyacrylamide gel electrophoresis PBCI2BI-BaPnd pRSVNeo-BP-The expression vectors pBC12BI-Bp and pRSVNeo-BO,containing full length BO fibrinogen chain cDNA, were prepared as previously described [12, 16]. All DNA fragments, obtained after restriction enzyme digestions, were purified by 1% agarose gel electrophoresis, electroelution, Immunoprecipitation of Nascent Fibrinogen Chains.
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