Difference between Internal Ribosome Entry Sites and kozak sequences.
If you are building something to express two proteins and you want to have a high probability of similar expression of both proteins you might want to use a "translational slip" also known as a "2A sequence", which is derived from those crafty picronaviruses. Your upstream protein is cloned without a stop codon, is appended with this sequence (EGRGSLLTCGDVEENPG*P) and followed nby your second protein in frame. "Translational skipping" occurs between the Glycine and Proline such that there is no covalent bond formed between these residues thereby releasing the upstream protein and continuing to make the downstream protein. This results in similar levels of expression of the two. We have tested these along side of IRES sequences and have found the latter (even the optimal IRES sequences) to give poorer expression of the downstream proteins.
(see doi:10.1016/j.tibtech.2005.12.006, doi:10.1186/1741-7007-6-40, doi:10.1038/nbt957 for review and examples)
IRES sequences in mRNAs can be recognized by ribosomes so the cap scanning is skipped.
If you are building something to express two proteins and you want to have a high probability of similar expression of both proteins you might want to use a "translational slip" also known as a "2A sequence", which is derived from those crafty picronaviruses. Your upstream protein is cloned without a stop codon, is appended with this sequence (EGRGSLLTCGDVEENPG*P) and followed nby your second protein in frame. "Translational skipping" occurs between the Glycine and Proline such that there is no covalent bond formed between these residues thereby releasing the upstream protein and continuing to make the downstream protein. This results in similar levels of expression of the two. We have tested these along side of IRES sequences and have found the latter (even the optimal IRES sequences) to give poorer expression of the downstream proteins.
(see doi:10.1016/j.tibtech.2005.12.006, doi:10.1186/1741-7007-6-40, doi:10.1038/nbt957 for review and examples)
I have a plasmid (pGADT7) containing my gene of interes (GOI) downstream of the T7 RNA polymerase. I wonder if I can express my GOI by transfecting this plasmid in a cell lign stably expressing the T7 RNA polymearase (BHK BSR-T7). Do you think it is possible please?
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