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Expression of functionally active human antithrombin III

Journal Article · · Proc. Natl. Acad. Sci. U.S.A.; (United States)
; ;
Human antithrombin III cDNA was cloned into an expression vector suitable for transient expression in COS cells. Upon transfection COS cells secreted a single immunoreactive 58-kDa protein. Quantitation of secretion levels by ELISA indicated that at 44 hr posttransfection cell sere secreting 48 +/- 5 ng of antithrombin III per 10/sup 6/ cells per 24 hr. Heparin-agarose chromatography resulted in the elution of the COS-derived protein as a broad band between 0.3 and 1.0 M NaC1. /sup 35/S-labeled medium from transfected cells reacted with human thrombin (1.5 ng/ml) in the absence of heparin. In 40 min, > 80% of the immunoreactive material was found as a higher molecular weight species, consistent with stoichiometric covalent complex formation. In a two-stage chromogenic thrombin inactivation assay, under pseudo-first-order conditions, at 16 nM antithrombin III the t/sub 1/2/ was 74 min and 50 min for plasma and COS cell-derived anithrombin III, respectively, in the absence of heparin. In the presence of 17.4 nM high-affinity heparin, the t/sub 1/2/ was 5.2 min and 2.2 min, respectively.
Research Organization:
Univ. of Colorado Health Sciences, Denver
OSTI ID:
5437254
Journal Information:
Proc. Natl. Acad. Sci. U.S.A.; (United States), Journal Name: Proc. Natl. Acad. Sci. U.S.A.; (United States) Vol. 84:12; ISSN PNASA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANTICOAGULANTS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CARBOHYDRATES
CLONING
COAGULANTS
DAYS LIVING RADIOISOTOPES
DNA
DNA-CLONING
DRUGS
ENZYME IMMUNOASSAY
ENZYMES
EVEN-ODD NUCLEI
GLUCOPROTEINS
HEMATOLOGIC AGENTS
HEMOSTATICS
HYDROLASES
IMMUNOASSAY
ISOTOPES
KINETICS
LIGHT NUCLEI
NUCLEI
NUCLEIC ACIDS
ORGANIC COMPOUNDS
PEPTIDE HYDROLASES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RECOMBINANT DNA
SACCHARIDES
SECRETION
SERINE PROTEINASES
SULFUR 35
SULFUR ISOTOPES
THROMBIN