I produced my protein( constructed in pet32b) in shuffl E coli and purified it by used of Ni-NTA column .
Now I want to removed the trx tag from our target protein.
If we do this with entrokinse enzyme, how can the next purification step be done?
Did you insert an entrokinse cleavage sequence in your expression construct? If there is a protease recognition site between Trx and your target protein, you can perform protease-mediated Trx cleavage. Once you cleave the Trx tag protein, you can separate your target protein from Trx in solution by size exclusion chromatography (SEC). Depending on the size of your target protein, you can use either Sephadex 200 or 75 column.
You can also purify your protein by using selective salting-out precipitation. However, purification by precipitation often depends upon the relative solubility of all the proteins in solution. I used Na2SO4 for selective salting-out precipitation of a recombinant protein from clarified cell lysates. You need to optimize the concentration of Na2SO4 that will precipitate your target proteins.
You can check the materials and methods section of the following papers. Here is the link to access the papers.
Article Integrated molecular and bioprocess engineering for bacteria...
Article Design strategies to address the effect of hydrophobic epito...
Hi Sebastian Schmitt
We used BamHI and HindIII.
Yes we have stop codon at the end of insert.
Hi Alemu Tekewe
we have entrokinase sequence in my construct.
Thank you very much for your advice.
Hi Sebastian Schmitt
Thank you so much for your help .
If we did not have the stop codon, how can done purification after digestion ?
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