Characterization and novel purification of recombinant human protein C from three mammalian cell lines.
Human Protein C (HPC), an antithrombotic factor with potential clinical utility, is a vitamin K-dependent protein that has several complex post-translational modifications. In an effort to define the functional roles of these modifications, recombinant HPC (rHPC) was expressed in and characterized from 3 adenovirus-transformed cell lines. The rHPC in crude culture medium from the 3 cell lines displayed anticoagulant activities that were either higher, slightly lower or much lower than that of plasma HPC. The rHPC from each cell line was purified and characterized using a novel, but simple chromatographic method, termed "pseudo-affinity", capable of resolving molecules differing by only very slight modifications. We demonstrate the critical dependence of full gamma-carboxylation on the function of this protein. In addition, our data indicate that both the gamma-carboxyglutamate and glycosyl contents affect the functional activities of rHPC.
Department of Biochemistry, Eli Lilly and Company, Indianapolis, IN 46285.
S C
SC
Yan
P
P
Razzano
Y B
YB
Chao
J D
JD
Walls
D T
DT
Berg
D B
DB
McClure
B W
BW
Grinnell
D000260:Adenoviruses, Human; D000818:Animals; D000925:Anticoagulants; D002241:Carbohydrates / Q000737:chemistry; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D002846:Chromatography, Affinity / Q000379:methods; D006224:Cricetinae; D005822:Genetic Vectors; D006801:Humans; D019158:N-Acetylneuraminic Acid; D010447:Peptide Hydrolases; D011486:Protein C / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012794:Sialic Acids / Q000378:metabolism; D014162:Transfection
D000925:Anticoagulants; D002241:Carbohydrates; D011486:Protein C; D011994:Recombinant Proteins; D012794:Sialic Acids; D010447:Peptide Hydrolases; D019158:N-Acetylneuraminic Acid