Research Paper | Published:

Characterization and Novel Purification of Recombinant Human Protein C from Three Mammalian Cell Lines

Bio/Technology volume 8, pages 655661 (1990) | Download Citation

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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 γ-carboxylation on the function of this protein. In addition, our data indicate that both the γ-carboxyglutamate and glycosyl contents affect the functional activities of rHPC.

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References

  1. 1.

    1989. The role of Protein C and thrombomodulin in the regulation of blood coagulation. J. Biol. Chem. 264: 4743–4746.

  2. 2.

    1988. The biochemistry of Protein C, p. 21–53. In: Protein C and Related Proteins. Bertina, R. M. (Ed.). Churchill Livingstone, New York.

  3. 3.

    1986. Report of workshop on expression of vitamin K- dependent proteins in bacterial and mammalian cells. Thrombosis Res. 44: 129–134.

  4. 4.

    , , , , , , , and 1987. Propeptide of human Protein C is necessary for gamma-carboxylation. Biochem. 26: 7003–7011.

  5. 5.

    , , , , , , and 1987. Vitamin K-dependent carboxylase: possible role of the substrate “Propeptide” as an intracellular recognition site. Proc. Natl. Acad. Sci. USA 84: 634–637.

  6. 6.

    , , , , , and 1989. Hydroxylation of aspartic acid in domains homologous to the epidermal growth factor precursor is catalysed by a 2-oxoglutarate-dependent dioxygenase. Proc. Natl. Acad. Sci. USA 86: 444–447.

  7. 7.

    , , , , , and 1989. Inhibitors of 2-ketoglutarate-deendent dioxygenase block aspartyl beta-hydroxylation of recombinant human Factor IX in several mammalian expression system. J. Biol. chem. 264: 6615–6618.

  8. 8.

    , , , , , and 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. Proc. Natl. Acad. Sci. USA 86: 3609–3613.

  9. 9.

    , , , , and 1988. Beta-hydroxyaspartic acid in the first epidermal growth factor-like domain of Protein C. J. Biol. Chem. 263: 19240–19248.

  10. 10.

    , , , , and 1988. The role of beta-hydroxyaspartate and adjacent carboxylate residues in the first EGF domain of human Factor IX. EMBO J. 7: 2053–2061.

  11. 11.

    and 1982. Amino acid sequence of the heavy chain of bovine Protein C. J. Biol. chem. 257: 12180–12190.

  12. 12.

    , , , , , , , , and 1986. Amino acid sequence of human von Willebrand Factor. Biochem. 25: 3171–3184.

  13. 13.

    , and 1989. Posttranslational modifications of proteins: some problems left to solve. Trends in Biological Sci. 14: 264–268.

  14. 14.

    , , and 1987. Transactivated expression of fully gamma-carboxylated recombinant human Protein C, an antithrombotic factor. Bio/Technology 5: 1189–1192.

  15. 15.

    , and 1988. A variant enhancer/regulatory region from a cloned human prototype BK virus genome. Nuc. Acids Res. 16: 9057.

  16. 16.

    , , , , and 1985. 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 cDNAs. Nuc. Acids Res. 13: 5233–5247.

  17. 17.

    , and 1986. Activation of the adenovirus and BK virus late promotors: Effects of the BK virus enhancer and trans-acting viral early proteins. Mol. Cell. Biol. 6: 3596–3605.

  18. 18.

    and 1983. An improved method for the determination of gamma-carboxyglutamic acid in proteins, bone and urine. Anal. Biochem. 131: 173–179.

  19. 19.

    , , and 1989. Formation and instability of o-phthalaldehyde derivatives of amino acids. Anal. Biochem. 178: 1–7.

  20. 20.

    , , , , , and 1987. Reversed-phase HPLC separation of dimethylaminoazobenzene sulfonyl and dimethylaminoazobenzene thiohydantoin-amino acid derivatives for amino acid analysis and microsequencing studies at the pmole level. Anal. Chem. 59: 1096–1101.

  21. 21.

    , and 1988. Monosaccharide analysis of glycoconjugates by anion exchange chromatography with pulsed amperometric detection. Anal. Biochem. 170: 54–62.

  22. 22.

    1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.

  23. 23.

    1976. A rapid and sensitive methhod for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.

  24. 24.

    1976. A new vitamin K-dependent protein. J. Biol. Chem. 251: 355–363.

  25. 25.

    , , and 1983. Structural changes required for activation of protein C are induced by Ca2+ binding to a high affinity site that does not contain gamma-carboxyglutamic acid. J. Biol. Chem. 258: 5554–5560.

  26. 26.

    and 1981. Protein C. Meth. Enzymol. 80: 320–332.

  27. 27.

    , , and 1985. Metal and phospholipid binding properties of partially carboxylated human prothrombin variants. J. Biol. Chem. 260: 9258–9264.

  28. 28.

    and 1982. Amino acid sequence of the light chain of bovine Protein C. J. Biol. Chem. 257: 12170–12179.

  29. 29.

    and 1988. Asparagine-linked oligosaccharides on lutropin, follitropin and thyrotropin. J. Biol. Chem. 263: 25–35.

  30. 30.

    , , , , , , and 1988. A 431 cell variants lacking the blood group A antigen display increased high affinity epidermal growth factor-receptor number, protein-tyrosine kinase activity, and receptor turnover. J. Cell Biol. 107: 939–949.

  31. 31.

    and 1988. Human beta-Protein C is not glycosylated at the fourth N-linked site. Blood 72: 371a.

  32. 32.

    and 1988. The effect of enzymatic removal of sialic acids on the functional properties of Protein C. Thrombosis & Haemostasis 60: 267–270.

  33. 33.

    , , , and 1986. Expression, purification and characterization of recombinant gamma-carboxylated Factor IX synthesized in Chinese hamster ovary cells. J. Biol. Chem. 261: 9622–9628.

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Author notes

    • S.C. Betty Yan

    Corresponding author.

Affiliations

  1. Departments of Biochemistry and Molecular Genetics, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285.

    • S.C. Betty Yan
    • , Pam Razzano
    • , Y. Bernice Chao
    • , Jenna D. Walls
    • , David T. Berg
    • , Don B. McClure
    •  & Brain W. Grinnell

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DOI

https://doi.org/10.1038/nbt0790-655

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