Expression and subcellular targeting of human insulin-like growth factor binding protein-3 in transgenic tobacco plants |
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Authors: | Stanley C K Cheung Samuel S M Sun Juliana C N Chan Peter C Y Tong |
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Institution: | (1) Department of Medicine and Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China;(2) Department of Biology, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China; |
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Abstract: | Human insulin-like growth factor binding protein-3 (hIGFBP-3) is a multifunctional protein which has high affinity for insulin-like
growth factor-I (IGF-I). It combines with IGF-I to form a tertiary complex in circulation, thus regulating the activity of
IGF-I. Furthermore, recombinant hIGFBP-3 (rhIGFBP-3) has been found to negatively regulate cell proliferation and induce apoptosis.
In this study, we have established an efficient plant bioreactor platform for mass production of rhIGFBP-3. Different expression
constructs, driven by the seed-specific phaseolin promoter, were designed and transformed into tobacco plant via Agrobacterium. To enhance protein expression level, the signal peptide (SP) and the C-terminal tetrapeptide AFVY of phaseolin were used
to direct rhIGFBP-3 to protein storage vacuole (PSV) in tobacco seed for stable accumulation. Western blot analysis showed
that rhIGFBP-3 was successfully synthesized in transgenic tobacco seeds, with the highest protein expression of 800 μg/g dry
weight. The localization of rhIGFBP-3 in PSV was also evident by confocal immunofluorescence microscopy. Our results indicated
that protein sorting sequences could benefit the expression level of rhIGFBP-3 and it is feasible to use plant as “bio-factory”
to produce therapeutic recombinant proteins in large quantity. |
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