Hi,
I want to purify a secreted protein by mammalian cells and I want it is tag-free.
So is there any possible to use the intein/chitin beads system like IMPACT system(NEB) to achieve this goal? I am concerning about the premature cleavage problems if I directly clone the intein and Chitin-binding domain to a eukaryotic expression vector like pCMV-HA.
Or do you have any suggestions to purify this secreted protein in mammalian cells without tag?
Thank you very much!
Since you already have the protein sequence and know about any subunits and glycosylation then certain purification characteristics such as pI can be determined by analysis programs. You may not need a tag. If a tag is your strategy then be cautious where it is placed in the sequence whether or not you use the intein approach or an enzyme cleaved conventional tag. You might want to stay away from the N- term for example in the case of processing within the cell. There are certain enzyme cleavage sequences that are better than others in generating the native termini.
I agree with Grant, enzyme cleavage sequences such as TEV or thrombin will be helpful.
Nevertheless, with the IMPACT system you will get a C-terminal thioester (in case you add a thiol to your solution). Do you want to have it, or do you want ot use the IMPACT system just for purification? It is rather problematic to hydrolyze your protein-thioester. If so, just introduce a His-Tag at the C-terminus and a cleavage site before, which can only be cleaved by non-mammalian proteases, so that your tag remains intact during the expression in the cells. Then you will be not in need of the IMPACT system. Hope this can be helpful.
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