I am interested in making a bicistronic system using viral 2A peptide to clone my protein of interest. The usual order of cloning is GOI-P2A-GFP (for example). Since the cleavage site is at the C terminus of the 2A peptide, is it okay to clone my GOI after the 2A so as to avoid the peptide tagging along with my protein (i.e., GFP-P2A-GOI)? The reason I ask this is because I have not seen any ready made vectors with such an arrangement.
The use of bicistronic vectors based on 2A implies that the first protein has 21 additional aa at the carboxy terminus and the second, 1 aa (Pro) in the amino terminus. If this does not affect the activity of the protein then the order can be anyone. Canvax Biotech supplies many expression vectors based in 2A.
The following paper may be relevant to your question:
J Biotechnol. 2014 Dec 20;192 Pt B:383-92. doi: 10.1016/j.jbiotec.2013.12.016.
Epub 2014 Jan 28.
Polycistronic expression of a β-carotene biosynthetic pathway in Saccharomyces
cerevisiae coupled to β-ionone production.
Beekwilder J(1), van Rossum HM(2), Koopman F(3), Sonntag F(4), Buchhaupt M(4),
Schrader J(4), Hall RD(5), Bosch D(6), Pronk JT(3), van Maris AJ(3), Daran JM(3).
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