Difference between revisions of "Team:Minnesota/2A Tags"
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<font size="3">• </font> 2A peptide sequences are ~20 amino acids in length and can easily be placed between genes of interest. When translated, the sequence causes the ribosome to skip over a peptide bond and allow the translation of multiple discrete polypeptides from a single mRNA molecule, leaving an 18 amino acid sequence on the C-terminus of the upstream protein and a proline residue on the N-terminus of the downstream protein (5). 2A viral sequences are particularly promising due to their small size (~60-70 nucleotides) and high cleavage rate which has been found to form 1:1 molar ratios of gene product in biscistronic sequences (3). These features are especially impressive when compared to internal ribosome entry sites (IRES), another popular method for creating polycistronic sequences in eukaryotes. IRES require large sequences (~500 nucleotides), which can be problematic when using size restricted vectors and can experience up to a 10-fold decrease in expression levels for downstream gene products (5). However, despite the attractiveness of using 2A sequences to create large multi-enzyme polycistronic sequences, little work has been done beyond simple bicistronic insertions and preliminary investigations suggest that the gene order within larger polycistronic sequences can affect the overall efficiency of larger pathways (5). | <font size="3">• </font> 2A peptide sequences are ~20 amino acids in length and can easily be placed between genes of interest. When translated, the sequence causes the ribosome to skip over a peptide bond and allow the translation of multiple discrete polypeptides from a single mRNA molecule, leaving an 18 amino acid sequence on the C-terminus of the upstream protein and a proline residue on the N-terminus of the downstream protein (5). 2A viral sequences are particularly promising due to their small size (~60-70 nucleotides) and high cleavage rate which has been found to form 1:1 molar ratios of gene product in biscistronic sequences (3). These features are especially impressive when compared to internal ribosome entry sites (IRES), another popular method for creating polycistronic sequences in eukaryotes. IRES require large sequences (~500 nucleotides), which can be problematic when using size restricted vectors and can experience up to a 10-fold decrease in expression levels for downstream gene products (5). However, despite the attractiveness of using 2A sequences to create large multi-enzyme polycistronic sequences, little work has been done beyond simple bicistronic insertions and preliminary investigations suggest that the gene order within larger polycistronic sequences can affect the overall efficiency of larger pathways (5). | ||
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<i><u><font size="3">• </font>Experimental Approach</u></i> | <i><u><font size="3">• </font>Experimental Approach</u></i> | ||
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− | (1) | + | (1) Cregg, J. M.; Tolstorukov, I.; Kusari, A.; Sunga, J.; Madden, K.; Chappell, T., Expression in the yeast Pichia pastoris. Meth. Enzymol. 2009, 463:169-89 |
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− | (2) | + | (2) Cullen, B. R.; Lomedico, P. T.; Ju, G., Transcriptional interference in avian retroviruses - implications for the promoter insertion model of leukaemogenesis. Nature. 1984, 307(5948): 241-245 |
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− | (3) | + | (3) Donnelly, M. L. L.; Hughes, L. E.; Luke, G. A.; Mendoza, H.; ten Dam, E.; Gani, D.; Ryan, M. D., The “cleavage” activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occuring “2A-like” sequences. Journal General Virology, 2001, 82: 1027-1041 |
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− | (4) | + | (4) Emmerman, M.; Temin, H. M., Comparison of promoter suppression in avian and murine retrovirus vectors. Nucleic Acids Res. 1986, 14(23): 9381-9396 |
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− | (5) | + | (5) Geier, M.; Fauland, P.; Vogl, T.; Glieder, A., Compact multi-enzyme pathways in P. pastoris. Chem. Commun. 2014, 51, 1643-1646. |
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− | (6) | + | (6) Keasling, J. D.; Mendoza, A.; Baran, P. S., Synthesis: A constructive debate. Nature. 2012, 492: 188-189 |
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− | (7) | + | (7) Ryan, M. D.; Drew, J., Foot-and-mouth disease virus 2A oligopeptide mediated cleabage of an artificial polyprotein. EMBO J. 1994, 13(4): 928-933 |
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− | (8) | + | (8) Zhu, T.; Guo, M.; Sun, C.; Qian, J.; Zhuang, Y.; Chu, J.; Zhang S., A systematical investigation on the genetic stability of multi-copy Pichia pastoris strains. Biotechnol Lett. 2009, 31(5): 679-84 |
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Revision as of 23:14, 18 September 2015