Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Factor IX has been expressed to high levels within a recombinant host cell and the biologically active fraction subsequently purified to homogeneity for characterization. The coding sequence for Factor IX was inserted into a mammalian cell expression vector and transfected into dihydrofolate reductase-deficient Chinese hamster ovary cells. The integrated DNA was amplified to a high copy number by selection for increasingly higher expression levels of the marker gene, dihydrofolate reductase, contained within a co-transfected plasmid. Cloned cell lines secreting over 100 micrograms/ml Factor IX antigen and up to 1.5 microgram/ml native Factor IX antigen have been obtained. Expression of biologically active Factor IX was dependent on the presence of vitamin K in the culture media. The gamma-carboxylated Factor IX was isolated from cell culture fluid by immunoaffinity chromatography using antibodies conformation-specific for the metal-stabilized conformer of Factor IX. This conformation is dependent upon metal ions and gamma-carboxyglutamic acid. Purified recombinant Factor IX migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an electrophoretic mobility equivalent to plasma-derived Factor IX. The purified recombinant Factor IX demonstrated Factor IX coagulant activity, measured in Factor IX-deficient plasma, of 35-75 units/mg. Amino acid analysis of the alkaline hydrolysate of recombinant Factor IX demonstrated an average of 6-7 mol of gamma-carboxyglutamic acid per mol of Factor IX. NH2-terminal sequence analysis of the first 17 residues revealed equivalent amino acid sequences for both purified recombinant and plasma-derived Factor IX. The results represent the first purification and characterization of a biologically active, gamma-carboxylated vitamin K-dependent protein expressed in a recombinant DNA system.
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PMID:Expression, purification, and characterization of recombinant gamma-carboxylated factor IX synthesized in Chinese hamster ovary cells. 373 88