Expression of the human igf-1 in transgenic plastids

Abstract

A plastid transformation vector for a stably transforming a plastid genome, comprising, as operably-linked components, a first flanking sequence, a DNA sequence coding for synthetic insulin-like growth factor-1 (IGF-1s) (seek ID NO. 1) or a substantially homologous DNA sequence of IGF-1s, which is capable of expression in the plastid genome, and a second flanking sequence.

Description

FIELD OF THE INVENTION [0001] This application relates to the field of genetic engineering of plant plastid genomes, particularly chloroplast, vectors for transforming plastids, transformed plants, progeny of transformed plants, and to methods for transforming plastid genomes of plants to generate Human insulin Growth factor (IGF). BACKGROUND [0002] Insulin-like growth factor 1 (IGF-1) is an anabolic hormone produced in the liver that is stimulated by the growth hormone (GH). GH binds to the GH receptors on the hepatocyte cell membrane and triggers an unknown mechanism that synthesizes and releases IGF-1 into the blood. The normal levels of IGF-1 are between 120-400 ng / ml. IGF-1 is involved in the regulation of cell proliferation and differentiation of a wide variety of cell and tissue types, and plays an important role in tissue renewal and repair. Because of these important applications of IGF-1 in the body, people who suffer IGF-1 deficiency have many harmful side effects. For ex...

AI Technical Summary

Benefits of technology

[0027] Still another aspect of the invention is an isolated and purified IFN derived from a chloroplast which has been transformed with the vector described above. Another aspect provides for an orally administrable therapeutic human interferon recombinant IFN, which is suitable for oral administration to a mammal. Yet another aspect of the invent...

Problems solved by technology

Because of these important applications of IGF-1 in the body, people who suffer IGF-1 deficiency have many harmful side effects.

However, the IGF-1 treatment is very expensive, $30,000 / mg.

The main problem with this expression system is that E. coli cannot produce the mature IGF-1 because E. coli does not form disulfide bonds in the cytoplasm.

The cost of IGF-1 production increases when the protein is in the periplasm because it is harder to purify and the IGF-1 is not properly folded because the disulfide bonds were not formed.

A drawback of the plant systems is the low expression levels of recombinant proteins.

However, recombinant protein expression in plants by nuclear transformation have been dismally low, with most levels much less than the 1% of total soluble protein that is needed for commercial feasibility if the protein must be purified (Daniell et al., 2002).

Thus, it is possible to engineer plant cells to contain up to 20,000 copies of a particular gene of interest which can result in very high levels of foreign gene expression.

Such gene transfers could potentially result in the emergence of “superweeds” able to resist certain herbicides thereby undermining the benefits of GM crops (Daniell, 2002).

Therefore, a foreign gene introduced by genetic engineering of the chloroplast genome could not transfer to genetically compatible weeds.

In contrast, nuclear transformation experiments in higher plants frequently suffer from epigenetic gene-silencing mechanisms resulting in inconsistent and unstable gene expression or complete loss of transgenic activity (Hager and Bock, 2000).

Good recombinant systems are still not available for many human proteins that are expensive to purify or highly susceptible to proteolytic degradation.

Proteolytic degradation is another serious concern for industrial bioprocessing.

In addition, until the Applicant's discovery, all production vehicles (E. coli, nuclear plant genomes, etc. . . .) have failed to provide a cost effective and functional IGF, which can be orally administered without the side effects, i.e. human pathogens that are associated with the current production vehicles.

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