Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 Oct 1;90(19):8957–8961. doi: 10.1073/pnas.90.19.8957

Processing of a fusion protein by endoprotease in COS-1 cells for secretion of mature peptide by using a chimeric expression vector.

Y C Liu 1, M Kawagishi 1, T Mikayama 1, Y Inagaki 1, T Takeuchi 1, H Ohashi 1
PMCID: PMC47480  PMID: 8415638

Abstract

The subtilisin-related proprotein convertase furin is expressed in various mammalian tissues. Expecting that COS-1 cells have a furin-like endoprotease, we constructed a fusion expression vector for production of a recombinant foreign protein having no signal peptide or a protein in truncated form into secreted mature protein. A cDNA fragment encoding N-terminal procalcitonin (pro-CT) of human calcitonin precursor was inserted into the mammalian expression vector pME18S. We used PCR techniques to generate four kinds of cDNAs encoding the C terminus of the pro-CT with Arg residues at P4 (Arg-Xaa-Lys-Arg), P6 (Arg-Xaa-Xaa-Xaa-Lys-Arg), or both (Arg-Xaa-Arg-Xaa-Lys-Arg), in addition to the Lys-Arg motif at the cleavage site, in order to determine the conditions for efficient processing in nonendocrine cells, such as COS-1 cells. The cDNA coding for the Fc fragment of human immunoglobulin G1 was fused in-frame to the cDNA encoding pro-CT at its C terminus. Upon transfection of the chimeric plasmids into COS-1 cells, almost all of the fusion protein with the Arg residues at both P4 and P6 were processed into secreted Fc product, even without cotransfection of furin. These results indicate that COS-1 cells have a furin-like endoprotease and suggest that pro-CT, with the Arg residues at both P4 and P6, can be used as a carrier peptide for expression of a foreign protein having no signal peptide or a protein in truncated form in COS-1 cells.

Full text

PDF
8957

Images in this article

8959Fig. 4
on p.8959
8959Fig. 5
on p.8959

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barr P. J. Mammalian subtilisins: the long-sought dibasic processing endoproteases. Cell. 1991 Jul 12;66(1):1–3. doi: 10.1016/0092-8674(91)90129-m. [DOI] [PubMed] [Google Scholar]
  2. Benjannet S., Rondeau N., Day R., Chrétien M., Seidah N. G. PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3564–3568. doi: 10.1073/pnas.88.9.3564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bresnahan P. A., Leduc R., Thomas L., Thorner J., Gibson H. L., Brake A. J., Barr P. J., Thomas G. Human fur gene encodes a yeast KEX2-like endoprotease that cleaves pro-beta-NGF in vivo. J Cell Biol. 1990 Dec;111(6 Pt 2):2851–2859. doi: 10.1083/jcb.111.6.2851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burns D. M., Birnbaum R. S., Roos B. A. A neuroendocrine peptide derived from the amino-terminal half of rat procalcitonin. Mol Endocrinol. 1989 Jan;3(1):140–147. doi: 10.1210/mend-3-1-140. [DOI] [PubMed] [Google Scholar]
  5. Ellison J. W., Berson B. J., Hood L. E. The nucleotide sequence of a human immunoglobulin C gamma1 gene. Nucleic Acids Res. 1982 Jul 10;10(13):4071–4079. doi: 10.1093/nar/10.13.4071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fuller R. S., Brake A. J., Thorner J. Intracellular targeting and structural conservation of a prohormone-processing endoprotease. Science. 1989 Oct 27;246(4929):482–486. doi: 10.1126/science.2683070. [DOI] [PubMed] [Google Scholar]
  7. Hatsuzawa K., Hosaka M., Nakagawa T., Nagase M., Shoda A., Murakami K., Nakayama K. Structure and expression of mouse furin, a yeast Kex2-related protease. Lack of processing of coexpressed prorenin in GH4C1 cells. J Biol Chem. 1990 Dec 25;265(36):22075–22078. [PubMed] [Google Scholar]
  8. Hosaka M., Nagahama M., Kim W. S., Watanabe T., Hatsuzawa K., Ikemizu J., Murakami K., Nakayama K. Arg-X-Lys/Arg-Arg motif as a signal for precursor cleavage catalyzed by furin within the constitutive secretory pathway. J Biol Chem. 1991 Jul 5;266(19):12127–12130. [PubMed] [Google Scholar]
  9. Ikeda K., Posillico J. T., Boyle M. B., Webb A. C., Brown E. M., Broadus A. E. Microinjected Xenopus oocytes secrete mature, biologically active parathyroid hormone. Mol Endocrinol. 1989 Jul;3(7):1084–1089. doi: 10.1210/mend-3-7-1084. [DOI] [PubMed] [Google Scholar]
  10. Kiefer M. C., Tucker J. E., Joh R., Landsberg K. E., Saltman D., Barr P. J. Identification of a second human subtilisin-like protease gene in the fes/fps region of chromosome 15. DNA Cell Biol. 1991 Dec;10(10):757–769. doi: 10.1089/dna.1991.10.757. [DOI] [PubMed] [Google Scholar]
  11. Korner J., Chun J., O'Bryan L., Axel R. Prohormone processing in Xenopus oocytes: characterization of cleavage signals and cleavage enzymes. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11393–11397. doi: 10.1073/pnas.88.24.11393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Misumi Y., Oda K., Fujiwara T., Takami N., Tashiro K., Ikehara Y. Functional expression of furin demonstrating its intracellular localization and endoprotease activity for processing of proalbumin and complement pro-C3. J Biol Chem. 1991 Sep 5;266(25):16954–16959. [PubMed] [Google Scholar]
  13. Mizushima S., Nagata S. pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res. 1990 Sep 11;18(17):5322–5322. doi: 10.1093/nar/18.17.5322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Nakayama K., Kim W. S., Torii S., Hosaka M., Nakagawa T., Ikemizu J., Baba T., Murakami K. Identification of the fourth member of the mammalian endoprotease family homologous to the yeast Kex2 protease. Its testis-specific expression. J Biol Chem. 1992 Mar 25;267(9):5897–5900. [PubMed] [Google Scholar]
  15. Palade G. Intracellular aspects of the process of protein synthesis. Science. 1975 Aug 1;189(4200):347–358. doi: 10.1126/science.1096303. [DOI] [PubMed] [Google Scholar]
  16. Pöschl E., Lindley I., Hofer E., Seifert J. M., Brunowsky W., Besemer J. The structure of procalcitonin of the salmon as deduced from its cDNA sequence. FEBS Lett. 1987 Dec 21;226(1):96–100. doi: 10.1016/0014-5793(87)80558-6. [DOI] [PubMed] [Google Scholar]
  17. Rehemtulla A., Dorner A. J., Kaufman R. J. Regulation of PACE propeptide-processing activity: requirement for a post-endoplasmic reticulum compartment and autoproteolytic activation. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8235–8239. doi: 10.1073/pnas.89.17.8235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Schalken J. A., Roebroek A. J., Oomen P. P., Wagenaar S. S., Debruyne F. M., Bloemers H. P., Van de Ven W. J. fur gene expression as a discriminating marker for small cell and nonsmall cell lung carcinomas. J Clin Invest. 1987 Dec;80(6):1545–1549. doi: 10.1172/JCI113240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Seidah N. G., Gaspar L., Mion P., Marcinkiewicz M., Mbikay M., Chrétien M. cDNA sequence of two distinct pituitary proteins homologous to Kex2 and furin gene products: tissue-specific mRNAs encoding candidates for pro-hormone processing proteinases. DNA Cell Biol. 1990 Jul-Aug;9(6):415–424. doi: 10.1089/dna.1990.9.415. [DOI] [PubMed] [Google Scholar]
  20. Seidah N. G., Marcinkiewicz M., Benjannet S., Gaspar L., Beaubien G., Mattei M. G., Lazure C., Mbikay M., Chrétien M. Cloning and primary sequence of a mouse candidate prohormone convertase PC1 homologous to PC2, Furin, and Kex2: distinct chromosomal localization and messenger RNA distribution in brain and pituitary compared to PC2. Mol Endocrinol. 1991 Jan;5(1):111–122. doi: 10.1210/mend-5-1-111. [DOI] [PubMed] [Google Scholar]
  21. Smeekens S. P., Avruch A. S., LaMendola J., Chan S. J., Steiner D. F. Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):340–344. doi: 10.1073/pnas.88.2.340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Smeekens S. P., Montag A. G., Thomas G., Albiges-Rizo C., Carroll R., Benig M., Phillips L. A., Martin S., Ohagi S., Gardner P. Proinsulin processing by the subtilisin-related proprotein convertases furin, PC2, and PC3. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8822–8826. doi: 10.1073/pnas.89.18.8822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Smeekens S. P., Steiner D. F. Identification of a human insulinoma cDNA encoding a novel mammalian protein structurally related to the yeast dibasic processing protease Kex2. J Biol Chem. 1990 Feb 25;265(6):2997–3000. [PubMed] [Google Scholar]
  24. Steiner D. F., Smeekens S. P., Ohagi S., Chan S. J. The new enzymology of precursor processing endoproteases. J Biol Chem. 1992 Nov 25;267(33):23435–23438. [PubMed] [Google Scholar]
  25. Takebe Y., Seiki M., Fujisawa J., Hoy P., Yokota K., Arai K., Yoshida M., Arai N. SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol Cell Biol. 1988 Jan;8(1):466–472. doi: 10.1128/mcb.8.1.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wise R. J., Barr P. J., Wong P. A., Kiefer M. C., Brake A. J., Kaufman R. J. Expression of a human proprotein processing enzyme: correct cleavage of the von Willebrand factor precursor at a paired basic amino acid site. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9378–9382. doi: 10.1073/pnas.87.23.9378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. van den Ouweland A. M., van Duijnhoven H. L., Keizer G. D., Dorssers L. C., Van de Ven W. J. Structural homology between the human fur gene product and the subtilisin-like protease encoded by yeast KEX2. Nucleic Acids Res. 1990 Feb 11;18(3):664–664. doi: 10.1093/nar/18.3.664. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES