title: Enhanced TGFbeta1 maturation in high five cells coinfected with recombinant baculovirus encoding the convertase furin/pace: improved technology for the production of recombinant proproteins in insect cells.
abstract: One important limitation of the widely used insect baculovirus overexpression system is its inefficiency to properly process heterologous proteins which are initially biosynthesized as larger inactive precursor proteins. One example is transforming growth factor beta 1 (TGFbeta1), a 25-kDa homodimeric protein with pleiotropic functions. As many growth factors, the inactive TGFbeta1 precursor molecule needs to be proteolytically cleaved C-terminal to a basic sequence to yield the mature and active homodimer. In insect cells, a large proportion of overexpressed TGFbeta1 was found in an inactive precursor form suggesting that the levels of endogenous convertases are limiting for the production of mature and bioactive TGFbeta1 in this system. We have demonstrated that furin, a member of a novel family of mammalian prohormone convertases (PCs) can efficiently process TGFbeta1 precursor resulting in the production of the mature and active growth factor. Taking advantage of this observation, we have developed an improved overproduction system for TGFbeta1 by coexpressing prohTGFbeta1 and human furin convertase in High Five cells. Using this system, the production of mature active TGFbeta1 increased in a dose-dependent fashion reaching up to 7. 8-fold the amount obtained with the growth factor only. Thus, eliminating the rate-limiting step in recombinant TGFbeta1 production maximizes its processing efficiency and the yield of the mature active growth factor. Such simple and efficient technology could be useful for large scale production of other proproteins which undergo similar maturation processes and share furin recognition sequences at the junction between the proregion and the mature polypeptide.
authors: Department of Pediatrics, Immunology Division, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada, J1H 5N4.
authors: M H
authors: MH
authors: Laprise
authors: F
authors: F
authors: Grondin
authors: C M
authors: CM
authors: Dubois
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D045683:Furin; D005822:Genetic Vectors; D006801:Humans; D008565:Membrane Proteins; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D018411:Spodoptera; D013381:Subtilisins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D016212:Transforming Growth Factor beta / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D013381:Subtilisins; C468731:FURIN protein, human; D045683:Furin