Production Of Salmon Calcitonin And Human Insulin-like Growth Factor Ⅰ Via The Oleosin-fusion Technology

Plant-based production of biopharmaceuticals, which is also refered as Plant Bioreactor or Molecular Pharming, has become an important research area of plant genetic engineering. Currently, a number of plant-based expression systems have been developed to produce recombinant biopharmaceuticals. Among all the systems, Plant oleosin fusion protein expression system, with oleosin as the "carrier" of recombinant proteins, provides a unique route for the large-scale production of recombinant proteins in plants, as well as an efficient process for putification of the desired polyptptide. In this study, Salmon calcitonin (sCT), ratα-amidating enzyme (α-AE) and human insulin-like growth factorⅠ(hIGF-Ⅰ) have been produced in Arabidopsis thaliana using this technology.Calcitonin is a 32-amino-acid peptide hormone with an N-terminal disulfide bridge and a C-terminal prolineamide residue. It is a potent inhibitor of osteoclastic bone resorption and plays an important role in the clinical treatment of bone related diseases, such as Osteoporosis and Paget's disease. In this study, sCTgly and msCTgly genes were synthesized using plant codon bias rule based on the amino acid sequences of sCTgly and msCTgly, which are the a-amidating enzyme substrates of sCT and msCT (a designed sCT analogue with higher bioactivity). The sCTgly gene was fused to the 3'-end of Glycine max 24 kDa oleosin gene, under the control of Cauliflower mosaic virus 35S constitutive promoter, and the resulting vector was used to transform A. thaliana. The fusion protein was found mainly accumulated in transgenic seeds and was correctly targeted to the oil body membrane. The msCTgly gene was fused to the 3'-end of Brassica napus 20 kDa oleosin in the same manner. Seed-specific expression of the oleosin-msCTgly fusion was directed via a B. napus oleosin promoter, and the resulting vector was used to transform A. thaliana. The oleosin-msCTgly fusion protein accumulated to ca.1.32% of the total seed protein in transgenic seeds. The fusion protein was separated from the majority of other seed proteins by flotation centrifugation. Recombinant msCTgly was released from the oleosin-msCTgly by thrombin endoprotease treatment, and was further purified through High performance Liquid Chromatography (HPLC). The expression level of msCTgly in transgenic seeds reached 0.17% of total seed protein, which was 8 times higher than reported before. Recombinant calcitonin was found to be unamidated by mass spectrometry analysis.The C-terminal prolineamide residue is essential for the bioactivity of calcitonin. To try to produce biologically active calcitonin directly from plants, rat a-amidating enzyme (a-AE) gene was transformed into A. thaliana. Plants expressing both calcitonin and a-amidating enzyme were obtained by crossing, which provides a basis for further studying the amidation of calcitonin in vivo. The a-AE was also fused to an oleosin for targeting to the oil body, which can be used as an immobilization matrix. This study provides a new alternative for the amidation of calcitonin in vitro.Human insulin-like growth factor I (hIGF-I) is also an important therapeutic protein. In this study, hIGF-Ⅰwas expressed in both E. coli and A. thaliana. hIGF-I with a polyhistidine tag was accumulated in bacteria to 9.6% of total bacteria protein, with half of the expressed protein being in soluble form. The hIGF-I was fused to the C-terminal of A. thaliana 18.5 kDa Oleosin and the fusion gene driven by oleosin promoter was transferred into A. thaliana. The accumulation of Oleosin-hIGF-I fusion in transgenic seed was up to 1% total seed protein. hIGF-I was released form the fusion via the TEV{Tobacco etch virus) protease, and the expression level of hIGF-I was 0.23% of the total seed protein, which was 7 times higher than reported before.