Abstract
A plant expression platform with eukaryotic post-translational modification (PTM) machinery has many advantages compared to other protein expression systems. This promising technology is useful for the production of a variety of recombinant proteins including, therapeutic proteins, vaccine antigens, native additives, and industrial enzymes. However, plants lack some of the important PTMs, including furin processing, which limits this system for the production of certain mammalian complex proteins of therapeutic value. Furin is a ubiquitous proprotein convertase that is involved in the processing (activation) of a wide variety of precursor proteins, including blood coagulation factors, cell surface receptors, hormones and growth factors, viral envelope glycoproteins, etc. and plays a critical regulatory role in a wide variety of cellular events. In this study, we engineered the human furin gene for expression in plants and demonstrated the production of a functional active recombinant truncated human furin in N. benthamiana plant. We demonstrate that plant produced human furin is highly active both in vivo and in vitro and specifically cleaved the tested target proteins, Factor IX (FIX) and Protective Antigen (PA83). We also demonstrate that both, enzymatic deglycosylation and proteolytic processing of target proteins can be achieved in vivo by co-expression of deglycosylating and furin cleavage enzymes in a single cell to produce deglycosylated and furin processed target proteins. It is highly expected that this strategy will have many potential applications in pharmaceutical industry and can be used to produce safe and affordable therapeutic proteins, antibodies, and vaccines using a plant expression system.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Antigens, Bacterial / genetics
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Antigens, Bacterial / metabolism
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Bacterial Toxins / genetics
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Bacterial Toxins / metabolism
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Factor IX / genetics
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Factor IX / metabolism
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Furin / biosynthesis*
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Furin / genetics*
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Furin / metabolism
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Humans
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In Vitro Techniques
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Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / genetics
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Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / metabolism
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Mice
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Nicotiana / genetics*
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Nicotiana / metabolism*
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Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / genetics
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Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / metabolism
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Plants, Genetically Modified / genetics
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Plants, Genetically Modified / metabolism
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Proprotein Convertases / genetics
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Proprotein Convertases / metabolism
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Protein Engineering / methods
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Protein Precursors / genetics
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Protein Precursors / metabolism
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Protein Processing, Post-Translational
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Tumor Necrosis Factor Ligand Superfamily Member 13 / genetics
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Tumor Necrosis Factor Ligand Superfamily Member 13 / metabolism
Substances
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Antigens, Bacterial
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Bacterial Toxins
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Protein Precursors
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Recombinant Proteins
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Tnfsf13 protein, mouse
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Tumor Necrosis Factor Ligand Superfamily Member 13
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anthrax toxin
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Factor IX
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Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
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Proprotein Convertases
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FURIN protein, human
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Furin
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Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Grants and funding
This study was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) (
www.tubitak.gov.tr) through the projects coded 114Z863 and 115S077 to Tarlan Mamedov. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.