I have been trying to express a recombinant protein in tobacco plants. However, based on the immunoblot results, there seem to be an accumulation of recombinant proteins with the chloroplast targeting peptides still fused to them. I am not sure what is causing this accumulation but i suspect that there could be an inefficiency in the import signal provided by the transit peptide. I would like to know
1) Is it possible that this accumulation can be caused by the transit peptide?
2) is there other forms of transit peptide with better efficiency or is it possible to perform modifications on the transit peptide to improve the import signal?
3) If theres a literature/journal that report such incidence, could you kindly share them?
The large subunits of RuBisCo are coded in the chloroplast itself, while the small subunits are coded in the nucleus of the cell. So, genetic engineering researchers are interested to use the chloroplast targeting signal peptide (or transit peptide) of small subunit of RuBisCo to translocate the proteins that are expressed in cytosol into chloroplasts for storage aiming at increasing the protein yield. Full length or truncated sequences have been attempted which give dissimilar efficiency of protein localisation. Modification on the original sequences might enhance the protein transport efficiency. One of the amino acid sequences is “MASSMLSSAAVVATRASAAQASMVAPFTGLKSAASFPVTRKQNNLDITSIASNGGRVQCA” extracted from Gils M et al. 2008.
Gils M et al. 2008. A novel hybrid seed system for plants. Plant Biotechnol J. 6(3): 226-35. http://onlinelibrary.wiley.com/doi/10.1111/j.1467-7652.2007.00318.x/abstract
You can get more information about the protein transport into chloroplast from the following review paper:
Jarvis P, Robinson C. 2004. Mechanisms of protein import and routing in chloroplasts. Curr Biol. 14(24): 1064-77. http://www.sciencedirect.com/science/article/pii/S0960982204009388
Did you used the tobaco Tp sequences? Because in some species the import machinery may have specific signatures that influence the efficiency of import.
Any way, this article and the reference there in, may help you. http://www.sciencedirect.com/science/article/pii/S0022283610008284
Thanks for the articles. It has been useful. From what i gather, truncation of transit peptide may or may not affect the import competence. A rather older study reported that a 2 amino acid deletion at the C-terminal of transit peptide significantly caused import incompetence ( http://www.ncbi.nlm.nih.gov/pubmed/12354964 ). Hence, there might be a possibility of reduced efficiency when using truncated transit peptide. One way to find out would be to compare the import competence of recombinant protein fused to either truncated or native transit peptide.
Thanks Mauricio and Jovan for the useful articles. Definitely, this can be an useful way to check the import competence; a good use of established truncated and native transit peptides in expressing the protein of interest might greatly expedite your research progress. All the best.
In my experience there is a big difference between stable transgenic plants and transient expression. When we express transit peptide fusions transiently by Agrobacterium you can often see the protein also accumulating in the cytosol. However in stable transgenic plants expressing the same constructs this never occurred. So to answer your question it would be good to know how exactly you express your protein.
all the best
Martin
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