Which residues or sequence should be inserted at the cleavage site to improve controllable cleavage in C-terminal pTYB vectors (pTYB1,2,3 and 4)?
Gly, LeuGluGly, LeuGluProGly or GlyThrLeuGluGly have been found to improve controllable cleavage in the C-terminal pTYB vectors, although these may not be the optimal sequences for every protein. The desired sequence can be included in the reverse PCR primer, and either the SapI or SmaI sites may be used for cloning. For both pTYB1 and pTYB11 vectors only the SapI site should be used to clone the 3´ and 5´ end respectively, of the target gene. This strategy will result in the fusion of the target gene adjacent to the intein tag (and the cleavage site). The target protein can be purified without any extra non-native residues. The use of SapI site allows for the addition of amino acids favorable for cleavage(by engineering them into the primers).
Related Questions
- In the case of the C-terminal fusion vectors (pTYB1,2,3 and 4), which residues at the C-terminal of the target protein may inhibit cleavage or cause in vivo cleavage?
- Which residues or sequence should be inserted at the cleavage site to improve controllable cleavage in C-terminal pTYB vectors (pTYB1,2,3 and 4)?
- In the case of C-terminal fusion vectors, if the EcoRI site is used as the 3´ cloning site, which vector should be chosen?
