I am trying to purify a protein using thioredoxin tag. The clone I made is in pET32b vector having N-terminal thioredxin tag and C-terminal His-tag. I already checked the plasmid using restriction digestion. When I tried to purify the protein , I got a very thick band around 12 kDa(Thioredoxin) and two faint bands around 29 kDa and ~42kDa (maybe fused protein with Thioredoxin tag as My protein is of around 29kDa). I am thinking (based on the SDS gel picture) that somehow the tag is expressed well and my protein is not expressing? can anyone tell me why is that? and what can be done?
Have you sequenced your plasmid? It sounds like either your gene is out of frame with the thioredoxin or you've somehow got a stop codon between them. (Unless you have any reason to believe that the tag would be cleaved off)
I would highly recommend thoroughly sequencing at least the expression cassette of every plasmid you create or modify before using it. There are a lot of ways cloning can go wrong and a restriction digest test won't give you enough information.
Dear Swati Srivastava
I think that you have to perform a small IMAC purificaiton trials to check if the 2 bands at 29 and 42 KDa bind to the resin which may confirm you hypotesis. In this case probably you have digestion at the linker beetween Thioredoxin and you protein.
You can try to solve it by changing expression condition (eg reduce induction temperature) or E.coli strain (using a strain depleted of proteases. But i'm not sure that in your case, it is a big issue, since the his tag is at the C-term and i suspect that after purification you would like to remove the Thioredoxin. I'm right?
So try to understand if the 2 bands contains your protein of interest and if after purification are stable.
At the following link you can find some suggestion to perform small scale imac purifcation
https://proteocool.blogspot.com/2021/05/tips-of-week7small-scale-imac.html
for purification from E.coli cytoplasm, instead cell surnatant you can just change the first part of the protocoll by performing small scale cell lysis by sonication or a cell lisys agent (eg Cellytic or B-per) similarly to the following protocol
https://proteocool.blogspot.com/2021/05/tip-of-week-6-rapid-protein-expression.html
and afterward you can perform a small scale imac using 100ul of NiNTA or similar resins.
If you perfom cell lysis with B-per or Cellitic, i suggest to you to mix the surnatant 1:1 with IMAC binding buffer before load the sample into the coloumn
best regards
Manuele
Hi Swati,
Your research seems promising and excellent!
As per my understanding the low expression of your protein could be attributed to various factors, including weak promoter strength, transcription or translation inhibition, poor solubility, proteolytic degradation, or incorrect folding. To address these issues, consider using a stronger promoter, optimizing expression conditions, employing a different expression system, incorporating protease inhibitors, and verifying protein folding. For protein purification, utilize a nickel-affinity column for His-tag-based separation, employ a cleavage enzyme for thioredoxin tag removal, and perform size exclusion chromatography to eliminate contaminants.
Thanks and Goodluck!
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