title: Co-expression of the protease furin in Nicotiana benthamiana leads to efficient processing of latent transforming growth factor-β1 into a biologically active protein.
abstract: Transforming growth factor beta (TGF-β) is a signalling molecule that plays a key role in developmental and immunological processes in mammals. Three TGF-β isoforms exist in humans, and each isoform has unique therapeutic potential. Plants offer a platform for the production of recombinant proteins, which is cheap and easy to scale up and has a low risk of contamination with human pathogens. TGF-β3 has been produced in plants before using a chloroplast expression system. However, this strategy requires chemical refolding to obtain a biologically active protein. In this study, we investigated the possibility to transiently express active human TGF-β1 in Nicotiana benthamiana plants. We successfully expressed mature TGF-β1 in the absence of the latency-associated peptide (LAP) using different strategies, but the obtained proteins were inactive. Upon expression of LAP-TGF-β1, we were able to show that processing of the latent complex by a furin-like protease does not occur in planta. The use of a chitinase signal peptide enhanced the expression and secretion of LAP-TGF-β1, and co-expression of human furin enabled the proteolytic processing of latent TGF-β1. Engineering the plant post-translational machinery by co-expressing human furin also enhanced the accumulation of biologically active TGF-β1. This engineering step is quite remarkable, as furin requires multiple processing steps and correct localization within the secretory pathway to become active. Our data demonstrate that plants can be a suitable platform for the production of complex proteins that rely on specific proteolytic processing.
authors.affiliation: Laboratory of Nematology, Plant Sciences Department, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: Ruud H P
authors.initials: RH
authors.lastname: Wilbers
authors.affiliation: Laboratory of Nematology, Plant Sciences Department, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: Lotte B
authors.initials: LB
authors.lastname: Westerhof
authors.affiliation: Laboratory of Nematology, Plant Sciences Department, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: Debbie R
authors.initials: DR
authors.lastname: van Raaij
authors.affiliation: Laboratory of Nematology, Plant Sciences Department, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: Marloes
authors.initials: M
authors.lastname: van Adrichem
authors.affiliation: Laboratory of Nematology, Plant Sciences Department, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: Andreas D
authors.initials: AD
authors.lastname: Prakasa
authors.affiliation: Laboratory of Nematology, Plant Sciences Department, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: Jose L
authors.initials: JL
authors.lastname: Lozano-Torres
authors.affiliation: Laboratory of Nematology, Plant Sciences Department, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: Jaap
authors.initials: J
authors.lastname: Bakker
authors.affiliation: Laboratory of Nematology, Plant Sciences Department, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: Geert
authors.initials: G
authors.lastname: Smant
authors.affiliation: Laboratory of Nematology, Plant Sciences Department, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: Arjen
authors.initials: A
authors.lastname: Schots
mesh_terms: D000818:Animals; D002460:Cell Line; D004847:Epithelial Cells / Q000187:drug effects; D045683:Furin / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007137:Immunoglobulin alpha-Chains / Q000235:genetics / Q000378:metabolism; D008907:Mink; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D058849:Protein Refolding; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*
chemical_list: D007137:Immunoglobulin alpha-Chains; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D053773:Transforming Growth Factor beta1; D045683:Furin
keywords: Nicotiana benthamiana; codon optimization; furin; proteolytic processing; signal peptide; transforming growth factor β1
references.citation: J Immunol. 1998 Feb 15;160(4):1589-97
references.pmid: 9469414
references.citation: Nat Rev Nephrol. 2010 Nov;6(11):643-56
references.pmid: 20838416
references.citation: J Exp Med. 2003 Dec 15;198(12):1875-86
references.pmid: 14676299
references.citation: Plant Biotechnol J. 2014 Dec;12(9):1333-42
references.pmid: 25196296
references.citation: Nat Immunol. 2008 Dec;9(12):1347-55
references.pmid: 18997793
references.citation: J Biol Chem. 1992 Mar 15;267(8):5668-75
references.pmid: 1544940
references.citation: Annu Rev Immunol. 2006;24:99-146
references.pmid: 16551245
references.citation: Immunity. 2006 Feb;24(2):179-89
references.pmid: 16473830
references.citation: Protein Expr Purif. 2006 Nov;50(1):9-17
references.pmid: 16901717
references.citation: Biochemistry. 1993 Feb 2;32(4):1164-71
references.pmid: 8424943
references.citation: Plant J. 2003 Mar;33(5):949-56
references.pmid: 12609035
references.citation: Reproduction. 2006 Aug;132(2):179-90
references.pmid: 16885528
references.citation: Algorithms Mol Biol. 2011 Nov 24;6:26
references.pmid: 22115189
references.citation: Science. 1990 Mar 16;247(4948):1328-30
references.pmid: 2315700
references.citation: Nat Immunol. 2008 Dec;9(12):1341-6
references.pmid: 18931678
references.citation: Plant Mol Biol. 1996 Feb;30(4):781-93
references.pmid: 8624409
references.citation: Plant Cell. 1995 Jun;7(6):677-88
references.pmid: 7647561
references.citation: Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3645-50
references.pmid: 20133581
references.citation: Plant Physiol. 2011 Jun;156(2):741-55
references.pmid: 21474436
references.citation: Nat Rev Mol Cell Biol. 2002 Oct;3(10):753-66
references.pmid: 12360192
references.citation: J Immunol. 1999 Apr 15;162(8):4567-75
references.pmid: 10201996
references.citation: J Am Coll Nutr. 2004 Jun;23(3):220-6
references.pmid: 15190046
references.citation: Mol Endocrinol. 1992 Oct;6(10):1691-700
references.pmid: 1448117
references.citation: Nature. 1992 Jul 30;358(6385):430-4
references.pmid: 1641027
references.citation: N Engl J Med. 2000 May 4;342(18):1350-8
references.pmid: 10793168
references.citation: Anal Biochem. 1994 Feb 1;216(2):276-84
references.pmid: 8179182
references.citation: Curr Opin Cell Biol. 1992 Aug;4(4):581-6
references.pmid: 1419038
references.citation: Protein Expr Purif. 2004 Oct;37(2):265-72
references.pmid: 15358346
references.citation: Mol Endocrinol. 1989 Jul;3(7):1090-8
references.pmid: 2677679
references.citation: Annu Rev Plant Biol. 2008;59:191-223
references.pmid: 18257708
references.citation: Int J Biochem Cell Biol. 1997 Jan;29(1):63-78
references.pmid: 9076942
references.citation: J Biol Chem. 1995 May 5;270(18):10618-24
references.pmid: 7737999
references.citation: Science. 1992 Jul 17;257(5068):369-73
references.pmid: 1631557
references.citation: J Immunol. 2010 Sep 15;185(6):3529-35
references.pmid: 20720212
references.citation: J Cell Sci. 2003 Jan 15;116(Pt 2):217-24
references.pmid: 12482908
references.citation: Plant Biotechnol J. 2012 Jan;10(1):83-94
references.pmid: 21895943
references.citation: Pharmacol Ther. 2003 May;98(2):257-65
references.pmid: 12725873
references.citation: J Cell Biol. 1994 Dec;127(6 Pt 2):1829-42
references.pmid: 7806563
references.citation: Biochem J. 2003 Jan 15;369(Pt 2):311-8
references.pmid: 12358597
references.citation: Plant Biotechnol J. 2010 Jun;8(5):529-63
references.pmid: 20132515
references.citation: Nature. 1982 Feb 18;295(5850):616-8
references.pmid: 6799842
references.citation: PLoS One. 2012;7(10):e46460
references.pmid: 23049703
references.citation: Plant Biotechnol J. 2011 Jun;9(5):618-28
references.pmid: 21535357