Karen K. Oishi
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Since the 1980s, the use of human proteins to treat and cure diseases has comprised an increasingly significant sector
of the pharmaceutical industry. In 2003, an estimated $39 billion of protein therapeutics were sold in the US. In the next
ten years, domestic sales of protein pharmaceuticals are estimated to grow to $359 billion.1 While traditional drugs are chemically synthesized, living organisms produce therapeutic proteins. The current generation
of microbial and mammalian cell bioreactor-based systems will not be able to handle future demand. Expensive small-capacity
units are too big a financial burden to produce and operate in the quantities needed. Manufacturing systems that use plants
as bioreactors can meet the growing demand of the healthcare industry for therapeutic proteins at far lower cost.
Plant chloroplast transformation technology (CTT) can deliver quality proteins at low cost. Several of the advantages of this
production platform are listed in Table 1. CTT in tobacco is now being validated as we explain below.
Photo courtesy of the Kentucky Research and Development Center, University of Kentucky, Lexington, KY.
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Chloroplasts Green plant cells contain three distinct genetic entities; a single nuclear genome and two plastomic organelle genomes.2 These organelles are the chloroplast and the mitochondria, each with multiple copies of a circular double-stranded DNA genome.
Each organelle contains replication, transcription, translation and processing machinery that is functionally and physically
separate from the rest of the cell. Since the late 1980s the light-harvesting chloroplast has been the focus of intense genetic
engineering research, which has established that plants with transformed chloroplasts can potentially provide low-cost, stable,
quality proteins.3-8 The chloroplast is a metabolic organelle designed to synthesize abundant proteins for energy generation and plant growth and
development. The chloroplast's genome is a self-replicating circular double-stranded DNA molecule ranging in size from 110
to 220 kilobase pairs depending on the species of plant. In the majority of plants, the chloroplast genome has two inverted
regions.2 With up to 100 genomes per chloroplast and up to 100 chloroplasts per cell, the integration of a transgene through homologous
recombination into the inverted repeat region can generate up to 20,000 copies of the transgene per cell.
The chloroplast generates proteins in a manner similar to a prokaryotic cell, creating polycistronic messages and accepting
large DNA inserts with multiple genes and repetitive elements without inducing silencing pathways. The chloroplast has been
demonstrated to properly fold and assemble disulfide-bonded proteins, which can be sequestered away from cytosolic proteases
during growth and development.9 However similar to bacterial expression systems, chloroplasts do not have the post-translational processing machinery for
glycosylation of proteins.
Table 1. Chloroplast Transformation Technology Advantages
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Source of a Million Seeds As shown in Figure 1, single or multiple genes can be integrated and expressed in transformed chloroplast as polycistronic
mRNA. The translational regulatory sequence, or ribosome-binding site (RBS), then directs high levels of target protein synthesis.
Dr. Henry Daniell, the technical founder of Chlorogen, developed universal transformation vectors, which direct integration
of the transgene to the inverted repeated region of plant chloroplasts by homologous recombination.10 The chloroplast transformation-expression vector is introduced into the chloroplast by particle bombardment of plant tissue,
or polyethylene glycol treatment of protoplast.3,8 Transformation is initiated by transgene integration into a few copies of chloroplast genome within a single cell, followed
by antibiotic selection and transgene replication during the next 15 to 20 cell divisions, which results in a homogeneous
plastid genome population. Integration of the transgene expression cassette into one of the inverted repeat regions will initiate
a copy number correction resulting in the duplication and insertion of another transgene cassette into the second inverted
repeat region.
