Biological activity of recombinant prohormone precursor insulin-like growth factor-1B (IGF-1B) and the mature form of IGF-1 expressed in transgenic plants.
Creators: name: Panahi, Mitra.
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 2001
Subjects: subject: Biology, Botany.

description: Recombinant DNA technology was used in an effort to produce insulin-like growth factor-1 (IGF-1) in large amount for therapeutic purposes. Therapeutic potential of human recombinant IGF-1 stems from the fact that IGF-1 resembles insulin in many aspects. Therefore IGF-1 is considered as a promising agent in treatment of some types of diabetes, especially in cases of insulin receptor defects. To manufacture large amounts of IGF-1, others have expressed human IGF-1 in bacteria and yeast systems. In both those foreign host cells, most of the recombinant protein was unable to fold properly and often accumulated to form inactive inclusion bodies. Also, together with the important limitations of other expression systems like mammalian cell cultures and transgenic animals, plants remained to be studied as a potential transgenic production system of choice for the expression of human recombinant IGF-1. Transgenic plants are potentially one of the most economical systems for large scale production of recombinant proteins for industrial and pharmaceutical uses. Low production costs and possibility of administering transgenic plants orally are some of the advantages of plant-based production (i.e. "protein farming"). In view of these advantages, human IGF-1 was expressed in transgenic tobacco and transgenic rice. Furthermore, the role of IGF-1 precursor (IGF-1B) in growth and differentiation is not clear. Despite some reports concerning the mitogenic activity of some parts of the E-peptide domain of this protein, no recombinant form of the full-length prohormone had been reported. In this thesis, IGF-1B was expressed in transgenic tobacco plants and showed biological activity comparable to that of recombinant IGF-1. Enzyme-linked immunosorbent assay (ELISA) data from leaves of transgenic plants detected higher expressing plants. These plants producing higher recombinant IGF-1 and IGF-1B levels were used for further studies. Southern analysis of transgenic plants detected the expected DNA fragment corresponding to the foreign expression construct. The copy reconstruction experiments showed that in most of the transgenic plants one copy of the transgene was incorporated. PCR and RT-PCR experiment data indicated recombinant IGF-1 and IGF-1B transgenes and transcripts were present in transgenic plants but absent in non-transformed plants. Western blot conducted on protein extracts from leaves of transgenic plants confirmed the expression of the recombinant proteins as being the same size as the standards. Biological assays showed the mitogenic effects of recombinant IGF-1 expressed in transgenic plants on a human cell line (human neuroblastoma cell line SH-SY5Y). Addition of non-transgenic plant extracts to the assay containing the E. coli recombinant human IGF-1 resulted in decrease of the rate of proliferation of the cells. This suggests that some compound(s) in the non-transformed leaf extracts inhibits the activity of IGF-1 protein. These data suggest that human IGF-1 and IGF-1B produced and stored in plants were functional and maintained in an active conformation and can be used for therapeutic purposes. Such a cheap and abundant supply of the recombinant protein as well can now facilitate further research on the role of IGF-1B itself and its relationship to IGF-1.