High Yield Expression of Recombinant Proteins Requiring Proteolytic Maturation: Use of the Endoprotease Furin

  • M. Himmelspach
  • U. Schlokat
  • B. Plaimauer
  • F. G. Falkner
  • F. Dorner
Part of the Cell Engineering book series (CEEN, volume 1)


During biogenesis, secreted eucaryotic proteins may undergo multiple post-translational modifications. The most prominent modifications include glycosylation, phosphorylation, acetylation, sulfation, β-hydroxylation, γ-carboxylation and disulphide-bond formation. In addition, the generation of biologically active molecules may require post-translational proteolytic processing. In this case the protein is synthesised as an inactive precursor molecule which, beside the removal of the signal peptide upon translocation into the endoplasmic reticulum, undergoes additional proteolytic cleavage(s) before secretion. Such endoproteolytic processing events typically remove N-terminal propeptides and/or convert a single chain precursor into a mature heterodimeric form. Secretion of proteins is commonly accomplished by two alternate routes. Precursor cleavage of proteins secreted via the regulated pathway (‘regulated secretory proteins’) usually occurs C-terminal to paired basic amino acid residues, while precursor processing of secreted proteins which transit the constitutive secretory pathway (‘constitutive secretory proteins’) requires a more complex basic motif. In either case, the enzymes responsible for precursor cleavage at these motifs were found to be homologous to the bacterial subtilisin/yeast kexin endoproteases and termed the family of pro-protein convertases. Furin, the first mammalian member of this family, is ubiquitously expressed in all cell types examined and was found to process the precursor molecules of a wide variety of physiologically important proteins, such as hormones, growth factors, receptors, plasma proteins, viral envelope proteins and bacterial toxins.


Precursor Processing Immature Secretory Granule Constitutive Secretory Pathway Recombinant Human Factor Single Chain Precursor 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Anderson, E.D., VanSlyke, J.K., Thulin, CD., Jean, F., and Thomas, G. (1997) Activation of the furin endoprotease is a multiple-step process: requirements for acidification and internal propeptide cleavage. EMBO J. 16, 1508–1518.PubMedCrossRefGoogle Scholar
  2. Anson, D.S., Austen, D.E.G., and Brownlee, G.G. (1985) Expression of active human clotting factor IX from recombinant DNA clones in mammalian cells. Nature 315, 683–685.PubMedCrossRefGoogle Scholar
  3. Ayoubi, T.A.Y., Meulemans, S.M.P., Roebroek, A.J.M., and Van de Ven, W.J.M. (1996) Production of recombinant proteins in Chinese hamster ovary cells overexpressing the subtilisin-like proprotein converting enzyme furin. Mol. Biol. Rep. 23, 87–95.PubMedCrossRefGoogle Scholar
  4. Barr, P.J., Mason, O.B., Landsberg, K.E., Wong, P.A., Kiefer, M.C., and Brake, A.J. (1991) cDNA and gene structure for a human subtilisin-like protease with cleavage specificity for paired basic amino acid residues. DNA and Cell Biol. 10, 319–328.CrossRefGoogle Scholar
  5. Ballinger, M.D., Tom, J., and Wells, J.A. (1996) Furilisin: a variant of subtilisin BPN’ engineered for cleaving tribasic substrates. Biochemistry 35, 13579–13585.PubMedCrossRefGoogle Scholar
  6. Benjannet, S., Rondeau, N., Day, R., Chrétien, M., and Seidah, N.G. (1991) PCI and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues. Proc. Natl. Arad. Sci. USA 88, 3564–3568.CrossRefGoogle Scholar
  7. Bentley, A.K., Rees, DJ., Rizza, C, and Brownlee, G.G. (1986) Defective propeptide processing of blood clotting factor IX caused by mutation of arginine to glutamine at position-4. Cell 45, 343–348.PubMedCrossRefGoogle Scholar
  8. Bonthron, D.T., Handin, R.I., Kaufman, R.J., Wasley, L.C., Orr, E.C., Mitsock, L.M., Ewenstein, B., Loscalzo, J., Ginsburg, D., and Orkin, S.H. (1986) Structure of pre-pro-von Willebrand factor and its expression in heterologous cells. Nature 324, 270–273.PubMedCrossRefGoogle Scholar
  9. Bosshart, H., Humphrey, J., Deignan, E., Davidson, J., Drazba, J., Yuan, L., Oorschot, V., Peters, P.J., and Bonifacino, J.S. (1994) The cytoplasmic domain mediates localization of furin to the trans-Golgi network. En route to the endosomal/lysosomal system. J. Cell Biol. 126, 1157–1172.PubMedCrossRefGoogle Scholar
  10. Bravo, D.A., Gleason, J.B., Sanchez, R.I., Roth, R.A., and Fuller, R.S. (1994) Accurate and efficient cleavage of the human insulin proreceplor by the human proprotein-processing protease furin. J. Biol. Chem. 269, 25830–25837.PubMedGoogle Scholar
  11. Brennan, S.O., and Nakayama, K. (1994) Cleavage of proalbumin peptides by furin reveals unexpected restrictions at the P2 and P’1 sites. FEBS Lett. 347, 80–84PubMedCrossRefGoogle Scholar
  12. Bauerfeind, R., and Huttner, W.B. (1993) Biogenesis of constitutive secretory vesicles, secretory granules and synaptic vesicles. Curr. Opin. Cell Biol. 5, 628–635.PubMedCrossRefGoogle Scholar
  13. Busby, S., Kumar, A., Joseph, M., Halfpap, L., Insley, M., Berkner, K., Kurachi, K., and Woodbury, R. (1985) Expression of active human factor IX in transfected cells. Nature 316, 271–273.PubMedCrossRefGoogle Scholar
  14. Chanat, E., Weiss, U., Huttner, W.B., and Tooze, S.A. (1993) Reduction of the disulphide bond of chromogranin B (secretogranin I) in the trans-Golgi network causes its missorting to the constitutive secretory pathways. EMBO J. 12, 2159–2168.PubMedGoogle Scholar
  15. Cool, D.R., Fenger, M., Snell, C.R., and Loh, Y.P. (1995) Identification of the sorting signal motif within pro-opiomelanocortin for the regulated secretory pathway. J. Biol. Chem. 270, 8723–8729.PubMedCrossRefGoogle Scholar
  16. Cool, D.R., Normant, E., Shen, F., Chen, H.C., Pannell, L., Zhang, Y., and Loh, Y.P. (1997) Carboxypeptidase E is a regulated secretory pathway sorting receptor: genetic obliteration leads to endocrine disorders in Cpe(fat) mice. Cell 88, 73–83.PubMedCrossRefGoogle Scholar
  17. Creemers, J.W.M., Jackson, R.S., and Hutton, J.C. (1998) Molecular and cellular regulation of prohormone processing. Sem. Cell and Dev. Biol. 9, 3–10.CrossRefGoogle Scholar
  18. Creemers, J.W.M., Siezen, R.J., Roebroek, A.J.M., Ayoubi, T.A.Y., Huylebroeck, D., and Van de Ven, W.J.M. (1993) Modulation of furin-mediated proprotein processing activity by site-directed mutagenesis. J. Biol. Chem. 268, 21826–21834.PubMedGoogle Scholar
  19. Creemers, J.W.M., Vey, M., Schäfer, W., Ayoubi, T.A.Y., Roebroek, A.J.M., Klenk, H.-D., Garten, W., and Van de Ven, W.J.M. (1995) Endoproteolytic cleavage of its propeptide is a prerequisite for efficient transport of furin out of the endoplasmic reticulum. J. Biol. Chem. 270, 2695–2702.PubMedCrossRefGoogle Scholar
  20. De Bie, I., Marcinkiewicz, M., Malide, D., Lazure, C., Nakayama, K., Bendayan, M., and Seidah, N.G. (1996) The isoforms of proprotein convertase PC5 are sorted to different subcellular compartments. J. Cell Biol. 135, 1261–1275.PubMedCrossRefGoogle Scholar
  21. De la Salle, H., Altenburger, W., Elkaim, R., Dott, K., Dieterlé, A., Drillien, R., Cazenave, J.-P., Tolstoshev, P., and Lecocq, J.-P. (1985) Active γ-carboxylated human factor IX expressed using recombinant DNA techniques. Nature 316, 268–270.PubMedCrossRefGoogle Scholar
  22. Dittie, A.S., Thomas, L., Thomas, G., and Tooze, S.A. (1997) Interaction of furin in immature secretory granules from neuroendocrine cells with the AP-1 adaptor complex is modulated by casein kinase II phosphorylation. EMBO J. 16, 4859–4870.PubMedCrossRefGoogle Scholar
  23. Diuguid, D.L., Rabiet, M.J., Furie, B.C., Liebman, H.A., and Furie, B. (1986) Molecular basis of haemophilia B: a defective enzyme due to an unprocessed propeptide is caused by a point mutation in the factor IX precursor. Proc. Natl. Acad. Sci. USA 83, 5803–5807.PubMedCrossRefGoogle Scholar
  24. Drews, R., Paleyanda, R.K., Lee, T.K., Chang, R.R., Rehemtulla, A., Kaufman, R.J., Drohan, W.N., and Lubon, H. (1995) Proteolytic maturation of protein C upon engineering the mouse mammary gland to express furin. Proc. Natl. Acad. Sci. USA 92, 10462–10466.PubMedCrossRefGoogle Scholar
  25. Foster, D.C., Rudinski, M.S., Schach, B.G., Berkner, K.L., Kumar, A.A., Hagen, F.S., Sprecher, C.A., Insley, M.Y., and Davie, E.W. (1987) Propeptide of human protein C is necessary for gamma-carboxylation. Biochem. 26, 7003–7011.CrossRefGoogle Scholar
  26. Fischer, B., Mitterer, A., Schlokat, U., DenBouwmeesler, R., and Dorner, F. (1994) Structural analysis of recombinant von Willebrand factor: identification of hetero-and homo-dimers. FEBS Lett. 351, 345–348.PubMedCrossRefGoogle Scholar
  27. Fischer, B.E., Schlokat, U., Mitterer, A., Reiter, M., Mundt, W., Turecek, P.L., Schwarz, H.P., and Dorner, F. (1995) Structural analysis of recombinant von Willebrand factor produced at industrial scale fermentation of transformed CHO cells co-expressing recombinant furin. FEBS Lett. 375, 259–262.PubMedCrossRefGoogle Scholar
  28. Fischer, B.E., Schlokat, U., Reiter, M., Mundt, W., and Dorner, F. (1997) Biochemical and functional characterization of recombinant von Willebrand Factor produced at large scale. Cell. Mol. Life Sei. 53, 943–950.CrossRefGoogle Scholar
  29. Fuller, R.S., Brake, A., and Thorner, J. (1989) Yeast prohormone processing enzyme (KEX2 gene product) is a Ca2+-dependent serine protease. Proc. Natl. Acad. Sci. USA 86, 1434–1438.PubMedCrossRefGoogle Scholar
  30. Furlan, M. (1996) Von Willebrand factor: molecular size and functional activity. Ann. Hematol. 72, 341–348.PubMedCrossRefGoogle Scholar
  31. Galeffi, P., and Brownlee, G.G. (1987) The propeptide region of clotting factor IX is a signal for a vitamin K dependent carboxylase: evidence from protein engeneering of amino acid-4. Nuel. Acids Res. 15, 9505–9513.CrossRefGoogle Scholar
  32. Gensberg, K., Jan, S., and Matthews, G.M. (1998) Subtilisin-related serine proteases in the mammalian constitutive secretory pathway. Sem. Cell and Dev. Biol. 9, 11–17.CrossRefGoogle Scholar
  33. Gordon, V.M., Rehemtulla, A., and Leppla, S.H. (1997) A role for PACE4 in the proteolytic activation of anthrax toxin protective antigen. Infect. Immun. 65, 3370–3375.PubMedGoogle Scholar
  34. Gut, A., Kappeier, F., Hyka, N., Balda, M.S., Hauri, H.P., and Matter, K. (1998) Carbohydrate-mediated Golgi to cell surface transport and apical targeting of membrane proteins. EMBO J. 17, 1919–1929.PubMedCrossRefGoogle Scholar
  35. Hamilton, M.A., and Charlebois, T.S. (1997) Extracellular propeptide processing of recombinant human factor IX by a secreted form of the endoprotease, PACE, in M.J.T. Carrondo, B. Griffiths and J.L.P. Moreira (eds.), Animal Cell Technology, Kluwer Academic Publishers, pp. 495–501.Google Scholar
  36. Hatsuzawa, K., Murakami, K., and Nakayama, K. (1992A) Molecular and enzymatic properties of furin, a Kex2-like endoprotease involved in precursor cleavage at Arg-X-Lys/Arg-Arg sites. J. Biochem. 111, 296–301.PubMedGoogle Scholar
  37. Hatsuzawa, K., Nagahama, M., Takahashi, S., Takada, K., Murakami, K., and Nakayama, K. (1992) Purification and characterization of furin, a kex2-like processing endoprotease, produced in Chinese hamster ovary cells. J. Biol. Chem. 267, 16094–16099.PubMedGoogle Scholar
  38. Himmelspach, M., Schlokat, U., Pfleiderer, M., Fischer, B., Antoine, G., Falkner, F.G., and Dorner, F. Recombinant human factor X: high yield expression and maturation by furin mediated in vitro processing. Submitted.Google Scholar
  39. Hosaka, M., Nagahama, M., Kim, W.S., Watanabe, T., Hatsuzawa, K., Ikemizu, J., Murakami, K., and Nakayama, K. (1991) Arg-X-Lys/Arg-Arg motif as a signal for precursor cleavage catalyzed by furin within the constitutive secretory pathway. J. Biol. Chem. 266, 12127–12130.PubMedGoogle Scholar
  40. Jean, F., Boudreault, A., Basak, A., Seidah, N.G., and Lazure, C. (1995) Fluorescent peptidyl substrates as an aid in studying the substrate specificity of human prohormone convertase PC1 and human furin and designing a potent irreversible inhibitor. J. Biol. Chem. 270, 19225–19231.PubMedCrossRefGoogle Scholar
  41. Jones, B.G., Thomas, L., Molloy, S.S., Thulin, C.D., Fry, M.D., Walsh, K.A., and Thomas, G. (1995) Intracellular trafficking of furin is modulated by the phosphorylation state of a casein kinase II site in its cytoplasmic tail. EMBO J. 14, 5869–5883.PubMedGoogle Scholar
  42. Kaufman, R.J. (1998) Post-translational modifications required for coagulation factor secretion and function. Thromb. Haemost. 79, 1068–1079.PubMedGoogle Scholar
  43. Kaufman, R.J., Wasley, L.C., Furie, B.C., Furie, B., and Shoemaker, C.B. (1986) Expression, purification, and characterization of recombinant γ-carboxylated factor IX synthesized in chinese hamster ovary cells. J. Biol. Chem. 261, 9622–9628.PubMedGoogle Scholar
  44. Lankhof, H., Van Hoeij, M., Schiphorst, M.E., Bracke, M., Wu, Y.-P., Ijsseldijk, M.J.W., Vink, T., De Groot, P.G., and Sixma, J.J. (1996) A3 domain is essential for interaction of von Willebrand factor with collagen type III. Thromb. Haemost. 75, 950–958.PubMedGoogle Scholar
  45. Leduc, R., Molloy, S.S., Thorne, B.A., and Thomas, G. (1992) Activation of human furin precursor processing endoprotease occurs by an intramolecular autoproteolytic cleavage. J. Biol. Chem. 267, 14304–14308.PubMedGoogle Scholar
  46. Lee, T.K., Drohan, W.N., and Lubon, H. (1995) Proteolytic processing of human protein C in swine mammary gland. J. Biochem. 118, 81–87.PubMedGoogle Scholar
  47. Lethagen, S.R. (1995) Pathogenesis, clinical picture and treatment of von Willebrand’s disease. Annals of Medicine 27, 641–651.PubMedCrossRefGoogle Scholar
  48. Leyte, A., Voorberg, J., Van Schijndel, H.B., Duim, B., Pannekoek, H., and Van Mourik, J.A. (1991) The pro-polypeptide of von Willebrand factor is required for the formation of a functional factor VIII-binding site on mature von Willebrand factor. Biochem. J. 274, 257–261.PubMedGoogle Scholar
  49. Liu, G., Thomas, L., Warren, R.A., Enns, C.A., Cunningham, C.C., Hartwig, J.H., and Thomas, G. (1997) Cytoskeletal protein ABP-280 directs the intracellular trafficking of furin and modulates proprotein processing in the endocytic pathway. J. Cell Biol. 139, 1719–1733.PubMedCrossRefGoogle Scholar
  50. Lubon, H., Paleyanda, R.K., Velander, W.H., and Drohan, W.N. (1996) Blood proteins from transgenic animal bioreactors. Transfus. Med. Rev. 10, 131–143.PubMedCrossRefGoogle Scholar
  51. Lusson, J., Benjannet, S., Hamelin, J., Savaria, D., Chrétien, M., and Seidah, N.G. (1997) The integrity of the RRGDL sequence of the proprotein convertase PC1 is critical for its zymogen and C-terminal processing and for its cellular trafficking. Biochem. J. 326, 737–744.PubMedGoogle Scholar
  52. Malide, D., Seidah, N.G., Chrétien, M., and Bendayan, M. (1995) Electron microscopic immunocytochemical evidence for the involvement of the convertases PC1 and PC2 in the processing of proinsulin in pancreatic beta-cells. J. Histochem. Cytochem. 43, 11–19.PubMedGoogle Scholar
  53. Marks, M.S., Woodruff, L., Ohno, H., and Bonifacino, J.S. (1996) Protein targeting by tyrosine-and di-leucine-based signals: evidence for distinct saturable components. J. Cell Biol. 135, 341–354.PubMedCrossRefGoogle Scholar
  54. Meulien, P., Nishino, M., Mazurier, C., Dott, K., Pietu, G., Jorieux, S., Pavirani, A., Girma, J.P., Oufkir, D., Courtney, M., and Meyer, D. (1992) Processing and characterization of recombinant von Willebrand factor expressed in different cell types using a vaccinia virus vector. Thromb. Haemost. 67, 154–160.PubMedGoogle Scholar
  55. Meulien, P., Balland, A., Lepage, P., Mischler, F., Dott, K., Hauss, C., Grandgeorge, M., and Lecocq, J.P. (1990) Increased biological activity of a recombinant factor IX variant carrying alanine at position +1. Prot. Engineering 3, 629–633.CrossRefGoogle Scholar
  56. Molloy, S.S., Thomas, L., VanSlyke, J.K., Stenberg, P.E., and Thomas, G. (1994) Intracellular trafficking and activation of the furin proprotein convertase: localization to the TGN and recycling from the cell surface. EMBO J. 13, 18–33.PubMedGoogle Scholar
  57. Molloy, S.S., Bresnahan, P.A., Leppla, S.H., Kumpel, K.R., and Thomas, G. (1992) Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg and efficiently cleaves anthrax toxin protective antigen. J. Biol. Chem. 267, 16396–16402.PubMedGoogle Scholar
  58. Mori, K., Kii, S., Tsuji, A., Nagahama, M., Imamaki, A., Hayashi, K., Akamatsu, T., Nagamune, H., and Matsuda, Y. (1997) A novel human PACE4 isoform PACE4E is an active processing protease containing a hydrophobic cluster at the carboxy terminus. J. Biochem. (Tokyo) 121, 941–948.Google Scholar
  59. Nagahama, M., Ikemizu, J., Misumi, Y., Ikehara, Y., Murakami, K., and Nakayama, K. (1991) Evidence that differenciates between precursor cleavages at dibasic and Arg-X-Lys/Arg-Arg sites. J. Biochem. 110, 806–811.PubMedGoogle Scholar
  60. Nakagawa, T., Murakami, K., and Nakayama, K. (1993) Identification of an isoform with an extremely large Cys-rich region of PC6, a Kex2-like processing endoprotease. FEBS Lett. 327, 165–171.PubMedCrossRefGoogle Scholar
  61. Nakayama, K. (1997) Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins. Biochem. J. 327, 625–635.PubMedGoogle Scholar
  62. Nakayama, K., Watanabe, T., Nakagawa, T., Kim, W.-S., Nagahama, M., Hosaka, M., Hatsuzawa, K., Kondoh-Hashiba, K., and Murakami, K. (1992) Consensus sequence for precursor processing at mono-arginyl sites. J. Biol. Chem. 267, 16335–16340.PubMedGoogle Scholar
  63. Normant, E., and Loh, Y.P. (1998) Depletion of carboxypeptidase E, a regulated secretory pathway sorting receptor, causes misrouting and constitutive secretion of proinsulin and proenkephalin, but not chromogranin A. Endocrinology 139, 2137–2145.PubMedCrossRefGoogle Scholar
  64. Oda, K., Misumi, Y., Ikehara, Y., Brennan, S.O., Hatzuzawa, K., and Nakayama, K. (1992) Proteolytic cleavages of proalbumin and complement pro-C3 in vitro by a truncated soluble form of furin, a mammalian homologue of the yeast Kex2 protease. Biochem. Biophys. Res. Comm. 189, 1353–1361.PubMedCrossRefGoogle Scholar
  65. Ohno, H., Fournier, M.C., Poy, G., and Bonifacino, J.S. (1996) Structural determinants of interaction of tyrosine-based sorting signals with the adaptor medium chains. J. Biol. Chem. 271, 29009–29015.PubMedCrossRefGoogle Scholar
  66. Orci, L., Ravazzola, M., Amherdt, M., Madsen, O., Vassalli, J.D., and Perrelet, A. (1985) Direct identification of prohormone conversion site in insulin-secreting cells. Cell 42, 671–681.PubMedCrossRefGoogle Scholar
  67. Paleyanda, R.K., Drews, R., Lee, T.K., and Lubon, H. (1997) Secretion of human furin into mouse milk. J. Biol Chem. 272, 15270–15274.PubMedCrossRefGoogle Scholar
  68. Preininger, A., Schlokat, U., Mohr, G., Himmelspach, M., Stichler, V., Kyd-Rebenburg, A., Plaimauer, B., Turecek, P.L., Schwarz, H.P., Falkner, F.G., Fischer, B.E., and Dorner, F. (1998) Strategies for recombinant furin employment in a biotechnological process: complete target protein precursor cleavage. Cytotechnology In press.Google Scholar
  69. Rehemtulla, A., Dorner, A.J., and Kaufman, R.J. (1992) Regulation of PACE propeptide-processing activity: requirement for a post-endoplasmic reticulum compartment and autoproteolytic activation. Proc. Natl Acad. Sci. USA 89, 8235–8239.PubMedCrossRefGoogle Scholar
  70. Rehemtulla, A., and Kaufman, R.J. (1992A) Preferred sequence requirements for cleavage of pro-von Willebrand factor by propeptide-processing enzymes. Blood 79, 2349–2355.PubMedGoogle Scholar
  71. Roebroek, A.J.M., Creemers, J.W.M., Ayoubi, T.A.Y., and Van de Ven, W.J.M. (1994) Furin-mediated proprotein processing activity: involvement of negatively charged amino acid residues in the substrate binding region. Biochimie 76, 210–216.PubMedCrossRefGoogle Scholar
  72. Roebroek, A.J.M., Schalken, J.A., Bussemakers, M.J.G., van Heerikhuizen, H., Onnekink, C., Debruyne, F.M.J., Bloemers, H.P.J., and Van de Ven, W.J.M. (1986) Characterization of human c-fes/fps reveals a new transcription unit (fur) in the immediately upstream region of the proto-oncogene. Mol. Biol. Rep. 11, 117–125.PubMedCrossRefGoogle Scholar
  73. Rouillé, Y., Duguay, S. J., Lund, K., Furuta, M., Gong, Q., Lipkind, G., Oliva Jr., A.A., Chan, S.J., and Steiner, D.F. (1995) Proteolytic processing mechanisms in the biosynthesis of neuroendocrine peptides: the subtilisin-like proprotein convertases. Front. Neuroendocrinol. 16, 322–361.PubMedCrossRefGoogle Scholar
  74. Rouillé, Y., Westermark, G., Martin, S.K., and Steiner, D.F. (1994) Proglucagon is processed to glucagon by prohormone convertase PC2 in αTCl-6 cells. Proc. Natl. Acad. Sci. USA 91, 3242–3246.PubMedCrossRefGoogle Scholar
  75. Rudolph, A.E., Mullane, M.P., Porche-Sorbet, R., and Miletich, J.P. (1997) Expression, purification and characterization of recombinant human factor X. Prot. Express. Purif. 10, 373–378.CrossRefGoogle Scholar
  76. Schäfer, W., Stroh, A., Berghofer, S., Seiler, J., Vey, M., Kruse, M.L., Kern, H.F., Klenk, H.D., and Garten, W. (1995) Two independent targeting signals in the cytoplasmic domain determine trans-Golgi network localization and endosomal trafficking of the proprotein convertase furin. EMBO J. 14, 2424–2435.PubMedGoogle Scholar
  77. Scheiffele, P., Peranen, J., and Simons, K. (1995) N-glycans as apical sorting signals in epithelial cells. Nature 378, 96–98.PubMedCrossRefGoogle Scholar
  78. Schlokat, U., Fischer, B.E., Herlitschka, S., Antoine, G., Preininger, A., Mohr, G., Himmelspach, M., Kistner, O., Falkner, F.G., and Dorner, F. (1996) Production of highly homogeneous and structurally intact recombinant von Willebrand Factor multimers by furin-mediated propeptide removal in vitro. Biotechnol. Appl. Biochem. 24, 257–267.PubMedGoogle Scholar
  79. Schlokat, U., Himmelspach, M., Falkner, F.G., and Dorner, F. (1997) Permanent gene expression in mammalian cells: gene transfer and selection, in H. Hauser and R. Wagner (eds.), Mammalian Cell Biotechnology in Protein Production, Walter de Gruyter, Berlin, New York, pp. 33–52.Google Scholar
  80. Seidah, N.G., Chrétien, M., and Day, R. (1994) The family of subtilisin/kexin like pro-protein and pro-hormone convertases: divergent or shared functions. Biochimie 76, 197–209.PubMedCrossRefGoogle Scholar
  81. Seidah, N.G., Day, R., Hamelin, J., Gaspar, A., Collard, M.W., and Chrétien, M. (1992) Testicular expression of PC4 in the rat: Molecular diversity of a novel germ cell-specific Kex2/subtilisin-like proprotein convertase. Mol. Endocrinol. 6, 1559–1570.PubMedCrossRefGoogle Scholar
  82. Seidah, N.G., Day, R., Marcinkiewicz, M., and Chrétien, M. (1998) Precursor convertases: An evolutionary ancient, cell-specific, combinatorial mechanism yielding diverse bioactive peptides and proteins. Ann NY Acad. Sci. 839, 9–24.PubMedCrossRefGoogle Scholar
  83. Seidah, N.G., Hamelin, J., Mamarbachi, M., Dong, W., Tadros, H., Mbikay, M., Chrétien, M., and Day, R. (1996) cDNA structure, tissue distribution, and chromosomal localization of rat PC7, a novel mammalian proprotein convertase closest to yeast kexin-like proteinases. Proc. Natl. Acad. Sci. USA 93, 3388–3393.PubMedCrossRefGoogle Scholar
  84. Siezen, R.J., Creemers, J.W.M., and Van de Ven, W.J.M. (1994) Homology modelling of the catalytic domain of human furin. A model for the eucaryotic subtilisin-like proprotein convertases. Eur. J. Biochem. 222, 255–266.PubMedCrossRefGoogle Scholar
  85. Smeekens, S.P., Avruch, A.S., LaMendola, J., Chan, S.J., and Steiner, D.F. (1991) Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans. Proc. Natl. Acad. Sci. USA 88, 340–344.PubMedCrossRefGoogle Scholar
  86. Smeekens S.P., Montag, A.G., Thomas, G., Albiges-Rizo, C., Carroll, R., Benig, M., Phillips, L.A., Martin, S., Ohagi, S., Gardner, P. et al. (1992) Proinsulin processing by the subtilisin-related proprotein convertases furin, PC2, and PC3. Proc. Natl. Acad. Sci. USA 89, 8822–8826.PubMedCrossRefGoogle Scholar
  87. Smeekens, S.P., and Steiner, D.F. (1990) Identification of a human insulinoma cDNA encoding a novel mammalian protein structurally related to the yeast dibasic processing protease Kex2. J. Biol. Chem. 265, 2997–3000.PubMedGoogle Scholar
  88. Spence, M.J., Sucic, J.F., Foley, B.T., and Moehring, T.J. (1995) Analysis of mutations in alleles of the fur gene from an endoprotease-deficient Chinese hamster ovary cell strain. Somat. Cell Mol. Gen. 21, 1–18.CrossRefGoogle Scholar
  89. Tanaka, S., Kurabuchi, S., Mochida, H., Kato, T., Takahashi, S., Watanabe, T., and Nakayama, K. (1996) Immunocytochemical localization of prohormone convertases PC1/PC3 and PC2 in rat pancreatic islets. Arch. Histol. Cytol. 59, 261–271.PubMedGoogle Scholar
  90. Takahashi, S., Hatsuzawa, K., Watanabe, T., Murakami, K., and Nakayama, K. (1994) Sequence requirements for endoproteolytic processing of precursor proteins by furin: transfection and in vitro experiments. J. Biochem. 116, 47–52.PubMedGoogle Scholar
  91. Takahashi, S., Nakagawa, T., Banno, T., Watanabe, T., Murakami, K., and Nakayama, K. (1995) Localization of furin to the trans-Golgi network and recycling from the cell surface involves Ser and Tyr residues within the cytoplasmic domain. J. Biol. Chem. 270, 28397–28401.PubMedCrossRefGoogle Scholar
  92. Takahashi, S., Nakagawa, T., Kasai, K., Banno, T., Duguay, S.J., Van de Ven, W.J.M., Murakami, K., and Nakayama, K. (1995A) A second mutant allele of furin in the processing-incompetent cell line, LoVo. J. Biol. Chem. 270, 26565–26569.PubMedCrossRefGoogle Scholar
  93. Tsuji, A., Hine, C., Tamai, Y., Yonemoto, K., Mori, K., Yoshida, S., Bando, M., Sakai, E., Mori, K., Akamatsu, T., and Matsuda, Y. (1997) Genomic organization and alternative splicing of human PACE4 (SPC4), kexin-like processing endoprotease. J. Biochem. 122, 438–452.PubMedGoogle Scholar
  94. Urbé, S., Tooze, S.A., and Barr, F.A. (1997) Formation of secretory vesicles in the biosynthetic pathway. Biochem. Biophys. Acta 1358, 6–22.PubMedCrossRefGoogle Scholar
  95. Van de Loo, J.W., Creemers, J.W., Bright, N.A., Young, B.D., Roebroek, A.J., and Van de Ven, W.J. (1997) Biosynthesis, distinct post-translational modifications, and functional characterization of lymphoma proprotein convertase. J. Biol. Chem. 272, 27116–27123.PubMedCrossRefGoogle Scholar
  96. Van den Ouweland, A.M.W., van Groningen, J.J.M., Roebroek, A.J.M., Onnekink, C., and Van de Ven, W.J.M. (1989) Nucleotide sequence analysis of the human fur gene. Nucleic Acids Res. 17, 7101–7102.PubMedCrossRefGoogle Scholar
  97. Van de Ven, W.J.M., Voorberg, J., Fontijn, R., Pannekoek, H., van den Ouweland, A.M.W., van Duijnhoven, H.L.P., Roebroek, A.J.M., and Siezen, R.J. (1990) Furin is a subtilisin-like pro-protein processing enzyme in higher eucaryotes. Mol. Biol. Rep. 14, 265–275.PubMedCrossRefGoogle Scholar
  98. Velander, W.H., Johnson, J.L., Page, R.L., Russell, C.G., Subramanian, A., Wilkins, T.D., Gwazdauskas, F.C., Pittius, C., and Drohan, W.N.(1992) High-level expression of a heterologous protein in the milk of transgenic swine using the cDNA encoding human protein C. Proc. Natl. Acad. Sci. USA 89, 12003–12007.PubMedCrossRefGoogle Scholar
  99. Velander, W.H., Page, R.L., Morcol, T., Russell, C.G., Canseco, R., Young, J.M., Drohan, W.N., Gwazdauskas, F.C., Wilkins, T.D., and Johnson, J.L. (1992A) Production of biologically active protein C in the milk of transgenic mice. Ann. NY Acad. Sci. 665, 391–403.PubMedCrossRefGoogle Scholar
  100. Vermeer, C. (1990) γ-Carboxyglutamate-containing proteins and the vitamin K-dependent carboxylase. Biochem. J. 266, 625–636.PubMedGoogle Scholar
  101. Verweij, C.L., Hart, M., and Pannekoek, H. (1987) Expression of variant von Willebrand factor (vWF) cDNA in heterologous cells: requirement of the pro-polypeptide in vWF multimer formation. EMBO J. 6, 2885–2890.PubMedGoogle Scholar
  102. Vey, M., Schäfer, W., Berghöfer, S., Klenk, H.D., and Garten, W. (1994) Maturation of the trans-Golgi network protease furin: compartmentalization of propeptide removal, substrate cleavage, and COOH-terminal truncation. J. Cell Biol. 127, 1829–1842.PubMedCrossRefGoogle Scholar
  103. Voorhees, P., Deignan, E., van Donselaar, E., Humphrey, J., Marks, M.S., Peters, P.J., and Bonifacino, J.S. (1995) An acidic sequence within the cytoplasmic domain of furin functions as a determinant of trans-Golgi network localization and internalization from the cell surface. EMBO J. 14, 4961–4975.PubMedGoogle Scholar
  104. Vidricaire, G., Denault, J.B., and Leduc, R. (1993) Characterization of a secreted form of human furin endoprotease. Biochem. Biophys. Res. Commun. 195, 1011–1018.PubMedCrossRefGoogle Scholar
  105. Watanabe, T., Murakami, K., and Nakayama, K. (1993) Positional and additive effects of basic amino acids on processing of precursor proteins within the constitutive secretory pathway. FEBS Lett. 320, 215–218.PubMedCrossRefGoogle Scholar
  106. Walter, P., and Johnson, A.E. (1994) Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane. Annu. Rev. Cell Biol. 10, 87–119.PubMedCrossRefGoogle Scholar
  107. Ware, J., Diuguid, D.L., Liebman, H.A., Rabiet, M.-J., Kasper, C.K., Furie, B.C., Furie, B., and Stafford, D.W. (1989). Factor IX San Dimas. J. Biol. Chem. 264, 11401–11406.PubMedGoogle Scholar
  108. Wasley, L.C., Rehemtulla, A., Bristol, J.A., and Kaufman, R.J. (1993) PACE/Furin can process the vitamin K-dependent pro-factor IX precursor within the secretory pathway. J. Biol. Chem. 268, 8458–8465.PubMedGoogle Scholar
  109. Watzke, H.H., and High, K.A. (1995) Factor X, in K.A. High and H.R. Roberts (eds.), Molecular Basis of Thrombosis and Hemostasis, Marcel Dekker, Inc., pp 239–255.Google Scholar
  110. Wise, R.J., Barr, P.J., Wong, P.A., Kiefer, M.C., Brake, A.J., and Kaufman, R.J. (1990) Expression of a human proprotein processing enzyme: correct cleavage of the von Willebrand factor precursor at a paired basic amino acid site. Proc. Natl. Arad. Sci. USA 87, 9378–9382.CrossRefGoogle Scholar
  111. Wise, R.J., Dorner, A.J., Krane, M., Pittman, D.D., and Kaufman, R.J. (1991) The role of von Willebrand factor multimers and propeptide cleavage in binding and stabilization of factor VIII. J. Biol. Chem. 266, 21948–21955.PubMedGoogle Scholar
  112. Wolf, D.L., Sinha, U., Hancock, T.E., Lin, P.-H., Messier, T.L., Esmon, C.T., and Church, W.R. (1991) Design of constructs for the expression of biologically active recombinant human factors X and Xa. J. Biol. Chem. 266, 13726–13730.PubMedGoogle Scholar
  113. Xu, H., and Shields, D. (1994) Prosomatostatin processing in permeabilized cells. J. Biol. Chem. 269, 22875–22881.PubMedGoogle Scholar
  114. Zhong, M., Benjannet, S., Lazure, C., Munzer, S., and Seidah, N.G. (1996) Functional analysis of human PACE4-A and PACE4-C isoforms: identification of a new PACE4-CS isoform. FEBS Lett 396, 31–36.PubMedCrossRefGoogle Scholar
  115. Zhou, A., Martin, S., Lipkind, G., LaMendola, J., and Steiner, D.F. (1998) Regulatory roles of the P domain of the subtilisin-like prohormone convertases. J. Biol. Chem. 273, 11107–11114.PubMedCrossRefGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1999

Authors and Affiliations

  • M. Himmelspach
    • 1
  • U. Schlokat
    • 1
  • B. Plaimauer
    • 1
  • F. G. Falkner
    • 1
  • F. Dorner
    • 1
  1. 1.Division of Baxter Inc.Biomédical Research Center, Hyland ImmunoOrth/DonauAustria

Personalised recommendations