Characterization of recombinant human antithrombin III synthesized in Chinese hamster ovary cells.
Biochemical and physiochemical properties of recombinant human antithrombin III were examined. This protein, produced in Chinese hamster ovary cells, showed a conformation apparently identical with the natural product isolated from human plasma when examined by circular dichroism, UV absorbance, and fluorescence spectroscopy. Comparison of the NH2-terminal sequences of recombinant and human plasma-derived antithrombin III showed that on synthesis and secretion of the recombinant protein from Chinese hamster ovary cells the signal peptide is correctly cleaved by the corresponding endoplasmic signal peptidase. The recombinant antithrombin III has identical properties in heparin binding and biological activities as determined in vitro by two-dimensional immunoelectrophoresis, progressive inhibitor, and heparin cofactor assays. Analysis of the carbohydrate portion of recombinant antithrombin III synthesized in Chinese hamster ovary cells revealed glycosylation of the complex type. Characterization of the oligosaccharide chains present in the recombinant protein reveals three major fractions, A (20%), B (60%), and C (20%). Fraction A contains tri- and tetraantennary complex-type oligosaccharides, fraction B contains biantennary oligosaccharides, and fraction C partially truncated biantennary structures. Pharmacokinetic studies with recombinant and plasma-derived antithrombin III in rabbits showed that the clearance behavior of both proteins is very similar and can be described by a double exponential decrease with almost identical kinetic parameters.
Zettlmeissl
G
G
Research Laboratories of Behringwerke AG, Marburg, Federal Republic of Germany.
Conradt
H S
HS
Nimtz
M
M
Karges
H E
HE
D000818:Animals; D000990:Antithrombin III / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D002241:Carbohydrates / Q000032:analysis; D002460:Cell Line; D002850:Chromatography, Gel; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D006493:Heparin / Q000378:metabolism; D006801:Humans; D007123:Immunoelectrophoresis, Two-Dimensional; D009844:Oligosaccharides / Q000302:isolation & purification; D010053:Ovary; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology
D002241:Carbohydrates; D009844:Oligosaccharides; D011994:Recombinant Proteins; D000990:Antithrombin III; D006493:Heparin