ABSTRACT
The production of therapeutic proteins in plant seed augments alternative production platforms such as microbial fermentation, cell-based systems, transgenic animals, and other recombinant plant production systems to meet increasing demands for the existing biologics, drugs under evaluation, and undiscovered therapeutics in the future. We have developed upstream purification technologies for oilseeds which are designed to cost-effectively purify therapeutic proteins amenable to conventional downstream manufacture. A very useful tool in these endeavors is the plant model system Arabidopsis thaliana. The current chapter describes the rationale and methods used to over-express potential therapeutic products in A. thaliana seed for evaluation and definitive insight into whether our production platform, Safflower, can be utilized for large-scale manufacture.
Subject(s)
Arabidopsis/genetics , Insulin , Plants, Genetically Modified , Recombinant Proteins , Agrobacterium , Bioreactors , Gene Expression , Genetic Vectors , Humans , Insulin/biosynthesis , Insulin/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/therapeutic use , Seeds/genetics , Transformation, GeneticABSTRACT
Gamma linolenic acid (GLA; C18:3Δ6,9,12 cis), also known as γ-Linolenic acid, is an important essential fatty acid precursor for the synthesis of very long chain polyunsaturated fatty acids and important pathways involved in human health. GLA is synthesized from linoleic acid (LA; C18:2Δ9,12 cis) by endoplasmic reticulum associated Δ6-desaturase activity. Currently sources of GLA are limited to a small number of plant species with poor agronomic properties, and therefore an economical and abundant commercial source of GLA in an existing crop is highly desirable. To this end, the seed oil of a high LA cultivated species of safflower (Carthamus tinctorius) was modified by transformation with Δ6-desaturase from Saprolegnia diclina resulting in levels exceeding 70% (v/v) of GLA. Levels around 50% (v/v) of GLA in seed oil was achieved when Δ12-/Δ6-desaturases from Mortierella alpina was over-expressed in safflower cultivars with either a high LA or high oleic (OA; C18:1Δ9 cis) background. The differences in the overall levels of GLA suggest the accumulation of the novel fatty acid was not limited by a lack of incorporation into the triacylgylcerol backbone (>66% GLA achieved), or correlated with gene dosage (GLA levels independent of gene copy number), but rather reflected the differences in Δ6-desaturase activity from the two sources. To date, these represent the highest accumulation levels of a newly introduced fatty acid in a transgenic crop. Events from these studies have been propagated and recently received FDA approval for commercialization as Sonova™400.
Subject(s)
Carthamus tinctorius/metabolism , Linoleoyl-CoA Desaturase/genetics , Saprolegnia/enzymology , Seeds/metabolism , gamma-Linolenic Acid/biosynthesis , Agrobacterium/genetics , Agrobacterium/metabolism , Carthamus tinctorius/genetics , Chemical Fractionation/methods , Culture Media/metabolism , Enzyme Activation , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Genes, Plant , Linoleoyl-CoA Desaturase/metabolism , Oleic Acid/metabolism , Phenotype , Plant Oils/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saprolegnia/genetics , Seeds/geneticsABSTRACT
Apolipoprotein AI Milano (ApoAI(Milano) ) was expressed as a fusion protein in transgenic safflower seeds. High levels of expression corresponding to 7 g of ApoAI(Milano) per kilogram of seed have been identified in a line selected for commercialization. The ApoAI(Milano) fusion protein was extracted from seed using an oilbody-based process and matured in vitro prior to final purification. This yielded a Des-1,2-ApoAI(Milano) product which was confirmed by biochemical characterization including immunoreactivity against ApoAI antibodies, isoelectric point, N-terminal sequencing and electrospray mass spectrometry. Purified Des-1,2-ApoAI(Milano) readily associated with dimyristoylphosphatidylcholine in clearance assays comparable to Human ApoAI. Its biological activity was assessed by cholesterol efflux assays using Des-1,2-ApoAI(Milano) :1-palmitoyl-2-oleoyl phosphatidylcholine complexes in vitro and in vivo. This study has established that high levels of biologically functional ApoAI(Milano) can be produced using a plant-based expression system.
Subject(s)
Apolipoprotein A-I/genetics , Carthamus tinctorius/genetics , Plants, Genetically Modified/metabolism , Seeds/genetics , Animals , Apolipoprotein A-I/metabolism , Apolipoprotein A-I/pharmacology , Carthamus tinctorius/metabolism , Cholesterol/blood , Lipid Metabolism , Mice , Mice, Inbred C57BL , Phosphatidylcholines/genetics , Phosphatidylcholines/metabolism , Phosphatidylcholines/pharmacology , Recombinant Fusion Proteins/metabolism , Seeds/metabolismABSTRACT
The increased incidence of diabetes, coupled with the introduction of alternative delivery methods that rely on higher doses, is expected to result in a substantial escalation in the demand for affordable insulin in the future. Limitations in the capacity and economics of production will make it difficult for current manufacturing technologies to meet this demand. We have developed a novel expression and recovery technology for the economical manufacture of biopharmaceuticals from oilseeds. Using this technology, recombinant human precursor insulin was expressed in transgenic plants. Plant-derived insulin accumulates to significant levels in transgenic seed (0.13% total seed protein) and can be enzymatically treated in vitro to generate a product with a mass identical to that of the predicted product, DesB(30)-insulin. The biological activity of this product in vivo and in vitro was demonstrated using an insulin tolerance test in mice and phosphorylation assay performed in a mammalian cell culture system, respectively.
