Plant molecular farming (PMF), defined as the practice of using plants to produce human therapeutic proteins, has received worldwide interest. PMF has grown and advanced considerably over the past two decades. A number of therapeutic proteins have been produced in plants, some of which have been through pre-clinical or clinical trials and are close to commercialization. Plants have the potential to mass-produce pharmaceutical products with less cost than traditional methods. Tobacco-derived antibodies have been tested and used to combat the Ebola outbreak in Africa. Genetically engineered immunoadhesin (DPP4-Fc) produced in green plants has been shown to be able to bind to MERS-CoV (Middle East Respiratory Syndrome), preventing the virus from infecting lung cells. Biosafety concerns (such as pollen contamination and immunogenicity of plant-specific glycans) and costly downstream extraction and purification requirements, however, have hampered PMF production from moving from the laboratory to industrial application. In this review, the challenges and opportunities of PMF are discussed. Topics addressed include; transformation and expression systems, plant bioreactors, safety concerns, and various opportunities to produce topical applications and health supplements.

Natto, a fermented soybean product, has been consumed as a traditional food in Japan for thousands of years. Nattokinase (NK), a potent blood-clot dissolving protein used for the treatment of cardiovascular diseases, is produced by the bacterium Bacillus subtilis during the fermentation of soybeans to produce Natto. NK has been extensively studied in Japan, Korea, and China. Recently, the fibrinolytic (anti-clotting) capacity of NK has been recognized by Western medicine. The National Science Foundation in the United States has investigated and evaluated the safety of NK. NK is currently undergoing a clinical trial study (Phase II) in the USA for atherothrombotic prevention. Multiple NK genes have been cloned, characterized, and produced in various expression system studies. Recombinant technology represents a promising approach for the production of NK with high purity for its use in antithrombotic applications. This review covers the history, benefit, safety, and production of NK. Opportunities for utilizing plant systems for the large-scale production of NK, or for the production of edible plants that can be used to provide oral delivery of NK without extraction and purification are also discussed.

Earthworms have been used as a traditional medicine in China, Japan, and other Far East countries for thousands of years. Oral administration of dry earthworm powder is considered as a potent and effective supplement for supporting healthy blood circulation. Lumbrokinases are a group of enzymes that were isolated and purified from different species of earthworms. These enzymes are recognized as fibrinolytic agents that can be used to treat various conditions associated with thrombosis. Many lumbrokinase (LK) genes have been cloned and characterized. Advances in genetic technology have provided the ability to produce recombinant LK and have made it feasible to purify a single lumbrokinase enzyme for potential antithrombotic application. In this review, we focus on expression systems that can be used for lumbrokinase production. In particular, the advantages of using a transgenic plant system to produce edible lumbrokinase are described.

The mycobacteriophage large serine recombinase Bxb1 catalyzes site-specific recombination between its corresponding attP and attB recognition sites. Previously, we and others have shown that Bxb1 has catalytic activity in various eukaryotic species including Nicotiana tabacum, Schizosaccharomyces pombe, insects and mammalian cells.

Healing of burns is a complex process and very few effective treatments exist to facilitate the burn recovery process. Human acidic fibroblast growth factor 1 (FGF-1) plays an important role in a variety of biological processes, including angiogenesis, and tissue repair. Salvia miltiorrhiza is widely used in traditional Chinese medicine as an herb for the treatment of various diseases, including cardiovascular and cerebrovascular diseases, and traumatic injuries. We present that expression of FGF-1 in S. miltiorrhiza significantly accelerates the healing of burn wounds. The human fgf-1 gene was fused with a barley α-amylase signal peptide DNA sequence and driven by a 35S promoter for constitutive expression in transgenic S. miltiorrhiza plants. The highest yield of recombinant FGF-1 obtained from leaves of transgenic S. miltiorrhiza lines was 272 ng/fresh weight. Aqueous extracts from transgenic S. miltiorrhiza exhibited FGF-1 activity approximately 19.2-fold greater than that of the standard FGF-1. Compared to the standard FGF-1 or the extracts obtained from non-transgenic plants, it stimulated proliferation of Balb/c 3 T3 mouse fibroblast cells assessed with the standard MTT assay and promoted angiogenesis in the chicken embryo chorioallantoic membrane (CAM) assay. Topical application of the extract significantly accelerated the burn wound healing process. The product appears to retain the biological activity of both FGF-1 as well as the medicinal properties of the plant. The extracts from transgenic S. miltiorrhiza combines the therapeutic functions of FGF-1 and the medicinal plant, S. miltiorrhiza. Topical application of the product can reduce the costs associated with extraction, purification, and recovery.

Lumbrokinases, a group of fibrinolytic enzymes extracted from earthworm, have been widely used to prevent and treat various cardiovascular diseases. They specifically target fibrin to effectively degrade thrombi without major side effects. Plant expression systems are becoming potential alternative expression platforms for producing pharmaceutical proteins. In this work, a lumbrokinase (PI239) was produced from a plant system. Both wild‐type (WT) and plant codon‐optimized (OP) PI239 gene sequences were synthesized and cloned into a geminivirus‐based single‐vector DNA replicon system. Both vectors were independently expressed in tobacco (Nicotiana tabacum) leaves transiently by agroinfiltration. Overexpressed PI239 resulted in sudden tissue necrosis 3 days after infiltration. Remaining proteins were purified through His‐tag affinity chromatography and analyzed with SDS‐PAGE and Western blot methods. Purified PI239 successfully degraded artificial fibrin with relative activity of 13,400 U/mg when compared with commercial lumbrokinase product. In vitro tests demonstrated that plant‐derived PI239 dissolved human blood clots and that the plant expression system is capable of producing functional PI239.