Please use this identifier to cite or link to this item: http://scholarbank.nus.edu.sg/handle/10635/17030
Title: Expression of recombinant proteins in tobacco system
Authors: DORAISWAMY TAMILSELVI
Keywords: Extrachromosomal replication, BY2 cell culture, signal peptide efficiency, Vascular Endothelial Growth Factor (hVEGF165), human Angiopoietin1 (hAng1)
Issue Date: 28-Apr-2005
Source: DORAISWAMY TAMILSELVI (2005-04-28). Expression of recombinant proteins in tobacco system. ScholarBank@NUS Repository.
Abstract: Transgenic plants have emerged as one of the most economical systems for large-scale production of recombinant proteins for human and animal health care uses in a field now popularly called as a??molecular farminga??. There are several reports now on transgenics, transplastomes and transient production of recombinant proteins in plants with commercial plant-derived recombinant proteins in market. Despite these developments, each plant-recombinant protein production system has to be designed, established and evaluated separately to improve the yield. In this study, the expression of foreign proteins in tobacco was evaluated by applying alternative expression strategies rather than conventional transgenic plant approach. In the first part, a plant a?? E. coli shuttle vector derived from replicon of the Ageratum yellow vein virus (AYVV) were tested for extrachromosomal replication in the tobacco BY2 cell suspension system. Clonal cell lines retained the vectors for more than four months with no apparent rearrangements. In next part, stable transgenic cell lines were established, evaluated for intra and extracellular accumulation of recombinant proteins. The intracellular accumulation of GUS and S65CGFP proteins were high in the clonal cell lines. Secretion of S65CGFP was low in the medium. This led to investigate on signal peptide efficiency in targeting the foreign protein. Novel signal peptides were identified from the BY2 suspension cell cultures, and their efficiency tested in the transient expression system. The plant signal peptides tested had higher efficiency than the native human secretory signals in the accumulation of the Vascular Endothelial Growth Factor (hVEGF165), a secreted protein. In the final part, a rapid transient expression system was developed and tested for production of human Angiopoietin1 (hAng1). Both proteins accumulated in plant cells and were glycosylated. The expression of the full length Ang1 has not been shown previously, these studies will be useful for its production, functional studies or perhaps for therapeutic purposes. Taken together, the findings reported here will be useful for controlled, contained, consistent and efficient production of therapeutic proteins providing an alternative to the currently used eukaryotic production systems.
URI: http://scholarbank.nus.edu.sg/handle/10635/17030
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