Hi to all,
I have expressed and purified a 6x his tagged metallothioein like cysteine rich protein. By means of various techniques such as electrophoresis and NMR, I have realized that the protein is unfolded and aggregates. Furthermore, I have tested various experimental conditions in order to study this protein, but no significant changes have been detected.
Do you think that the enzymatic his-tag removal could change something?
Furthermore, this his tag is near to the cysteine rich motif, so I think that it could interfere with the folding of the protein (for example i imagine that the his tag could bind metals and bridge some cysteine), and this reason could support the his tag removal.
Thank you!
I definitely would try to remove the His-taq. Unfortunately I do not have a reference in mind, but some former colleagues and I observed negative effects of His-taq on protein folding.
It is also worth to clone the His-Taq on the other terminus of the protein and analyze the folding!
Does your protein has N-terminal his-tag or C-terminal?
If you have the Thrombin (or any other) cleavage site between tag and actual protein, you may try to cleave the tag. Let me know if you need any help for tag-removal.
Alternatively, you may express the tag at the other terminal as Marlen suggested. Some proteins which are misfolded because of N-terminal His tag, folds properly with C-terminal His-tag and vice versa.
Hi Antonio,
I also agree with the above responses and also there are several reports in the literature indicating that His-Tag can change properties of the protein and it may also interfere with the folding. However there can be other factors also affecting the folding. As indicated..you have tried several optimizations (howveevr they are not mentioned so one cannot comment on them)....the aggrgation many times is due to very high expresison of the protein in heterologous sytem which may favour undesirbale interactions resulting into aggregation. It might be prevented by several modification that either allow slow induction (some you may have already tried such as low temperature). You may also reduce the conc. of the inducer to express the proein slowly for a long time so that the local protein conc. is low and it is able fold properly. You may also bind the His-Tag to a Ni-NTA resin first and then try a on-column refolding approach (literature is available). In this cae the bound His-Tag will not interefere with the protein folding....some optimiztions should definitely work...You can also try cloning with vectors containg MBP tag which makes protein more soluble....
bye
Ajay
ok thanks to all, the tag is precisely at the c-term, and it's near to a cysteine rich motive supposed to be a heavy-matal binding domain, subsequently i'm inclined to believethat the tag could interact with the motif and some metal could bridge between the residues. so i think that by digesting the tag, something could change resulting in a improving of the protein folding.
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