Characterization and Novel Purification of Recombinant Human Protein C from Three Mammalian Cell Lines
- 1 July 1990
- journal article
- Published by Springer Nature in Nature Biotechnology
- Vol. 8 (7) , 655-661
- https://doi.org/10.1038/nbt0790-655
Abstract
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.Keywords
This publication has 19 references indexed in Scilit:
- Post-translational modifications of proteins: some problems left to solveTrends in Biochemical Sciences, 1989
- Aspartyl beta-hydroxylase: in vitro hydroxylation of a synthetic peptide based on the structure of the first growth factor-like domain of human factor IX.Proceedings of the National Academy of Sciences, 1989
- Inhibitors of 2-Ketoglutarate-dependent Dioxygenases Block Aspartyl β-Hydroxylation of Recombinant Human Factor IX in Several Mammalian Expression SystemsPublished by Elsevier ,1989
- Hydroxylation of aspartic acid in domains homologous to the epidermal growth factor precursor is catalyzed by a 2-oxoglutarate-dependent dioxygenase.Proceedings of the National Academy of Sciences, 1989
- A variant enhancer/regulatory region from a cloned human prototype BK virus genomeNucleic Acids Research, 1988
- Propeptide of human protein C is necessary for .gamma.-carboxylationBiochemistry, 1987
- Vitamin K-dependent carboxylase: possible role of the substrate "propeptide" as an intracellular recognition site.Proceedings of the National Academy of Sciences, 1987
- Report of workshop on expression of vitamin k-dependent proteins in bacterial and mammalian cells Madison, Wisconsin, USA, April, 1986Thrombosis Research, 1986
- Amino acid sequence of human von Willebrand factorBiochemistry, 1986
- The structure and evolution of a 461 amino acid human protein C precursor and its messenger RNA, based upon the DNA sequence of cloned human liver cDNAsNucleic Acids Research, 1985