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.
Highlights
ObjectivesThe major goal of this study was to achieve furin processing in plants and apply the technology to the post-translational modification (PTM) of proteins, in which furin cleavage is necessary for maturation and activation
We engineered the human furin gene for expression in N. benthamiana plants resulting in the production of a highly soluble, functionally active enzyme
Patients with hemophilia B are mainly treated with Factor IX (FIX), obtained from concentrates made from human blood and recombinant FIX produced in CHO cells
Summary
The major goal of this study was to achieve furin processing in plants and apply the technology to the PTM of proteins, in which furin cleavage is necessary for maturation and activation
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