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


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

Table 1. Development of alternative insulin delivery technologies
Until recently, insulin could only be effectively administered through injection; however, significant efforts have been made by a number of biotechnology and pharmaceutical companies to develop technologies that will allow insulin to be administered in other ways. Some companies working on the development of alternative delivery technologies are listed in Table 1.

For some developers of alternative delivery technologies, access to a low cost, high volume supply of insulin is key to the successful commercialization of their technology. Based on pharmacokinetic analysis, companies developing alternative insulin delivery technologies estimate that these technologies will require approximately 5 to 20 times more insulin than the amount required for injection. We project that with the commercialization of alternative delivery technologies, manufacturing de-mands will increase from the current 4,000 kg/yr to an estimated 16,000 kg/yr by 2010. We believe that our safflower-produced insulin will allow us to supply this expanding market while simultaneously reducing the cost of insulin production.

The current cost of insulin production is approximately $50 to 60 per gram. We believe our technology will allow us to reduce the unit cost by at least 40%. Based on information received from published sources, we believe the capital expenditure associated with building insulin-manufacturing facilities can cost over $250 million per ton of capacity. We project that our technology will reduce the capital cost of manufacturing facilities by 70%.


Figure 6. Schematic representation of human insulin
Insulin is a small, 5.8 kDa polypeptide hormone consisting of an A chain of 21 amino acids and a B chain of 30 amino acids linked together with two cysteine disulfide bridges (Figure 6).

As a first approach to demonstrating proof-of-principle for insulin production, we introduced human mini-insulin (Des-B30) into our model organism Arabidopsis thaliana.7 Des-B30 insulin is a biologically active form of human insulin that differs from authentic insulin by the deletion of the C-terminal threonine amino acid on the B chain. Des-B30 is produced in other transgenic systems (e.g., yeast) and this form of insulin can be matured to authenticity through a simple in vitro transpeptidation reaction.8 To optimize accumulation, genetic constructs were engineered to target expression of the recombinant insulin to both the endoplasmic reticulum (ERi) and the oilbody surface (OBi). In addition to the Des-B30 insulin gene and Trypsin-cleavable pro-peptide sequence, present in both the OBi and ERi constructs, the ERi construct included a KDEL (lysine-aspartate-glutamate-leucine) endoplasmic reticulum retention signal peptide and an affinity tag for oleosin.

Figure 7. Expression of Des-B30 insulin in transgenic oilseed. (A) Oilbody prepared proteins (20 μg) from seeds expressing the ERi (affinity capture technology) or OBi (Stratosome technology) transgene compared to non-recombinant (Wild type) oilbody proteins separated on 15% SDS-PAGE and Coomassie-stained; (B) the corresponding Western blot probed with anti-insulin monoclonal antibody E2E3 (ab9569; Abcam, Cambridge, Mass.). Symbols: (Arrows = fusion proteins; Wt = wild type seed; M = molecular marker)
Using the oilbody extraction and purification technology described above, recombinant insulin was purified and analyzed to quantify the expression of ERi and OBi (Figure 7). We were able to demonstrate through this analysis that Arabidopsis can express insulin at a level of 0.13% of total seed protein. Subsequent experiments conducted with variations of the ERi constructs have resulted in expression levels at commercially relevant levels of 1.15% of total seed protein. This expression level is approximately 50 times higher than previously reported transgenic plant systems engineered to produce insulin.9

Plant-Derived Insulin Can Be Matured In Vitro With Trypsin

To enable insulin maturation and recovery following oilbody separation, the Des-B30 insulin fusion protein was engineered to contain a trypsin cleavable pro-peptide. SemBioSys has shown that when recombinant mini-insulin is trypsin-cleaved from its fusion partner, it undergoes full protein maturation to the expected Des-B30 insulin. Insulin maturation and authenticity relative to commercial insulin standard was confirmed through mass spectral analysis (Figure 8).

Figure 8. Mass spectral analysis of plant-derived insulin. Human insulin standard (Sigma) in comparison to Des-B30 insulin derived from trypsin matured endoplasmic reticulum and oilbody surface seed. Samples were purified by reverse phase high-performance liquid chromatography prior to analysis.

The expected molecular mass of Des-B30 insulin is 5706.5 Da. The observed molecular mass of the trypsin-cleaved-matured OBi product (5706.30 Da) precisely matched the expected molecular mass and, therefore, directly corresponded to Des-B30 insulin. The difference between the expected and observed molecular mass for trypsin-cleaved-matured ERi (6191.51 Da) corresponded to a Des-B30 insulin with a KDEL ER retention peptide on the A chain of the cleaved product (Des-B30 insulin-KDEL).

Biological Equivalence to Commercial Insulin

Figure 9. Insulin Tolerance Test. Temporal changes in serum glucose levels in male B6 mice following intraperitoneal (IP) injection of insulin standards (Humulin R or Roche) in comparison to OBi derived Des-B30 insulin and negative controls (saline or identically treated non-recombinant OB derived fractions).
Using an established animal model we demonstrated the biological equivalence of plant-produced insulin relative to commercial varieties of insulin. OBi was used in an insulin tolerance test performed on 15 two-month-old C57/6 male mice. This bioassay was performed to determine the in vivo effect of SemBioSys's plant-derived insulin (Figure 9).


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