Protein targeting, transgene

Yang, J., Barr, L. A., Fahnestock, S. R., and Liu, Z. B. (2005). High yield recombinant silk-like protein production in transgenic plants through protein targeting. Transgenic Res. 14,313-324. 

A., Verger, R., Doherty, A., Merot, B. and Danzin, C. (2001) Large-scale production of a therapeutic protein in transgenic tobacco plants effect of subcellular targeting on quality of a recombinant dog gastric lipase. Molecular Breeding 7, 329-340. 

In principle, gene therapy is not limited to any particular spectrum of diseases. Gene therapy is limited by the type of compound that can be delivered, i.e., nucleic acids or their protein/peptide transgene products. The particular vector that carries this compound determines the target cell type in which it is expressed, as well as the... 

APL is most often associated with the t(15 17)(q22 q21) reciprocal chromosomal translocation which causes the fusion of the RARa locus with a gene of unknown function called PML (for promyelocytic leukaemia) and expression of PML-RARa chimeric proteins in all leukaemic cells [82-86]. Expression of the PML-RARa protein in transgenic mice results in development of APL which, as the human disease, can be induced into remission by ATRA treatment [87-90]. These results and studies with APL cells in vitro, or cells exogenously expressing PML-RARa, suggest that this oncoprotein is also a primary target of ATRA action [91, 92]. 

Little is known of how the biosynthetic metabolon is assembled, what mechanisms control the membrane-specific targeting, and how the conversions to apocarotenoids occur. Yet the current approach to drive import of bacterial or plant genes is to use transit sequences of a stromal protein that may limit the effectiveness of the transgene. In addition, for specific applications of controlling carotenoid composition, we need to better understand the interactions of the various enzymes,... 

The wild type ilvA gene was modified to target the protein to the plastid and expressed in A. thaliana. Transgenic plants showed a 20-fold increase in levels of 2-ketobutyrate as well as a large increase in 2-aminobutyrate, the transaminated product of 2-ketobutyrate [27, 41]. The levels of threonine remained stable whereas isoleucine concentration increased. Constitutive expression of the ilvA protein along with bktB, phaA, and phaC proteins in the plastids of A. thaliana led to the synthesis of poly(3HB-co-3HV) in the range of 0.2 - 0.8 % dry weight, with a HV level between 4-17 mol % [27,41]. Co-expression of the iso-... 

Fig. 1.3 Prediction of the most appropriate subcellular targeting strategies by agroinfiltration. The levels of an industrial enzyme (IE) are shown in agroinfiltrated and transgenic alfalfa leaves using different subcellular targeting peptides. Equal amounts of total soluble leaf proteins were separated by SDS-PAGE and blotted onto a PVDF membrane. Polyclonal anti-IE IgGs were used for detection.

Transcriptional inhibitors could be used simultaneously. Rifampicin blocks chloroplast and mitocondrian RNA synthesis [23, 24], while tagetitoxin is a very specific inhibitor of chloroplast RNA polymerase [25]. Treatment with these antibiotics does not inhibit Rubisco SSU synthesis since the promoter is part of the nuclear genome, while the cytosolic ribosomes are not affected by streptomycin. Therefore SSU promoters can be used to drive transgene expression and facilitate the accumulation of recombinant proteins. Expressed proteins are targeted to a suitable cellular compartment, such as the cytoplasm, apoplastic space or chloroplast, depending on the nature of the protein. 

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