Producing Proteins Using Transgenic Oilbody-Oleosin Technology - Progress has been significant in producing therapeutic proteins in plants. Insulin is an early candidate for commercialization. - BioPh

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Producing Proteins Using Transgenic Oilbody-Oleosin Technology
Progress has been significant in producing therapeutic proteins in plants. Insulin is an early candidate for commercialization.


BioPharm International
Volume 19, Issue 6

The Advantages of Seed Oilbodies

The oilbody-oleosin technology exploits oilseed plants, typically safflower, to produce recombinant proteins abundantly and at low cost. The technology is built on two basic proprietary capabilities. The first is the capability to express recombinant proteins in seed oilbodies. The second is the ability to extract oilbodies inexpensively from seeds.


Figure 1. Cross-section of a safflower seed stained to show proteins













The oilbody is a unique seed–oil storage organelle (Figure 1). Oil-bodies possess a highly organized structure consisting of a core of neutral lipid or triacylglycerol surrounded by a half-unit membrane of phospholipids and an outer surface layer composed primarily of a single class of protein known as oleosin. (Figure 2)


Figure 2. Oilbody structure and a detail showing location of the oleosin at the surface.














Oleosins are unique proteins with natural surfactant properties derived from alternating amphipathic and lipo-philic domains. The central domain of oleosins is highly lip-ophilic, containing the longest continuous stretch of hydrophobic amino acids yet reported, tightly anchoring the protein within the neutral lipid core of the oilbody. The amphipathic N- and C-termini interact with the phospholipids membrane forming a network that surrounds the oilbody in an amphipathic shell. The resulting structure is a highly stable, natural microparticle.


Figure 3. Picture A depicts an oleosin protein targeted to a natural oilbody. Picture B depicts the Stratosome Biologics System in which an oleosin–recombinant protein fusion (designated GeneX) is targeted to an oilbody to form a recombinant oilbody carrying the desired protein.






















Flexible Recombinant Protein Production


Figure 4. Flowchart of the Stratosome Biologics System
We have developed two proprietary recombinant protein production systems, the Stratosome Biologics System and the Affinity Capture System. Together, these systems offer the ability to produce a wide array of recombinant proteins. This technology uses genetic engineering to express recombinant proteins in oilbodies in the seed (Figure 3). It works at a large-scale using a continuous flow procedure (Figure 4).







The Stratosome Biologics System

Using plant genetic engineering techniques, we can target recombinant proteins to the surface of oilbodies through covalent fusions with oleosin. The combination of oilbody engineering with oilbody extraction is the basis of the Stratosome Biologics System. Here are the steps:

  • Genetic engineering is used to introduce a transgene encoding an oleosin–GeneX fusion protein into the plant. As the plant grows and the seed develops, the oleosin–GeneX fusion is expressed to produce a recombinant fusion protein such as insulin or apolipoprotein AI. The recom-binant fusion protein then targets the oilbody to form a recombinant oilbody, as depicted in Figure 3B.
  • Seed is produced from the genetically engineered plants using conventional farming practices that have been adapted to ensure product integrity and confinement.
  • The harvested seed is then processed using the proprietary oilbody extraction process depicted in Figure 4.
  • In cases where a purified protein is the end product, an enzyme or chemical that recognizes a cleavage site between the target protein and the oleosin is added to purified oilbodies. The enzyme or chemical then cleaves the recombinant protein from the oleosin.
  • The oilbodies are then removed via centrifugation and the recombinant protein can then be further purified further using conventional downstream processing techniques with simple steps and at low cost.

A wide array of proteins, ranging in size from ~1 kDa (nine amino acids) to over 100 kDA, have been produced using the Stratosome Biologics System. In addition to simple proteins, we have demonstrated that proteins with complex secondary and tertiary structure, including those requiring the formation of multiple disulfide bridges, can be produced using this system. We have achieved recombinant protein accumulation levels of more than 5% of total seed protein for some proteins, which is five times higher than our minimum commercial threshold.

Affinity Capture System

In cases where a protein does not accumulate as an oleosin fusion, for example with proteins which must be exposed to the secretory pathway, a modification is available that combines conventional plant protein expression with oleosin technology. In this modification, an affinity ligand is fused either to the oleosin or the protein of interest through genetic engineering. The desired protein can then be targeted for deposition in any cellular compartment. When the seed is ground up, the protein of interest associates with the oilbody and the resulting oilbody–protein assembly can be separated from the rest of the seed components. The desired protein is then disassociated from the oilbody using conventional techniques (changing pH or ionic strength for example), or, if the ligand was attached to the protein of interest, the fusion is cleaved. The protein of interest is then available for final purification.

The Extraction Process: Oil is Lighter Than Water


Figure 5. Oilbody extraction technology
We have developed technology to extract and stabilize intact oilbodies from seeds using a cost-effective aqueous extraction process. This process is depicted in Figure 5 and is based on the differential density of oil and water. The seeds are homogenized using a mill. The oilbodies are then separated from the balance of the seed material and washed using a series of centrifuges to remove seed-derived impurities. The resulting product consists of 75% oilbodies and 25% water, with negligible carryover of other seed materials. The vast majority of the seed proteins are removed by this process. At this stage, we have removed over 98% of the contaminating proteins originally present in the seed, and the resultant oilbody fraction is highly enriched with the recombinant target protein. Conventional downstream processing completes purification of the recombinant protein. Oilbody processing dramatically simplifies downstream processing and reduces the overall cost of bulk protein production by reducing the number of costly chromatographic steps.

We currently operate a 1,800-ft2 pilot plant capable of processing approximately 100 kg/hr of seed (43 kg/hr of oilbodies), and are working to establish a cGMP cabability to produce clinical quantities of our pharmaceutical protein product candidates for use in Phase 1 and Phase 2 clinical trials.

INSULIN:A PRODUCT CANDIDATE

We are developing insulin derived from genetically engineered safflower to serve the expanding diabetes market and to facilitate the commercialization of alternative insulin delivery technologies.

According to the World Health Organization, diabetes affected an estimated 177 million individuals worldwide in 2000 and is projected to affect over 370 million individuals by 2030.5 Insulin therapy is the exclusive treatment for Type 1 diabetics and is also being more frequently prescribed for Type 2 diabetics. In 2003, the worldwide insulin market was valued at approximately $4.3 billion and is projected to grow to $7.1 billion by 2010, due to growth in incidence, diagnosis, usage, and the commercialization of new insulin technologies.6


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