I am struggling with a Mycobacterium protein (phthalate esterase), which upon overexpression forms inclusion body (maybe) and it is found in the pellet fraction. I have tried cloning the protein with two different vectors but faced the same problem.
Vectors used - pET28a and pMAL-c2X (attached image below).
Can anyone suggest me on this?
How to achieve recovery from pellet fractions using denaturation/renaturation protocol?
Can denaturation / renaturation be done with fused protein (i.e. insert with pMAL-c2X)?
Looking forward for your suggestions
Thank you
Hello For this kind of question, I think you can try different ways from my experience.
First, using chaperone protein to increase the amount of soluable form of protein.
Second, optimize expression conditions.
Third, change vector,like pCOLD-TF.
Hope they can help you.
Best,
Yanli
Dear Mousami,
What is your objective for expressing the mycobacterial protein? If it is a non-toxic gene then it is not a problem. Even if your expressed protein is seen as inclusion bodies there are many ways to re-solubilize and renature the protein of interest. Initially try lowering the expression conditions, one factor at a time ((Temp:37, 25, 18), Time, rpm, and IPTG concentration (if it is an inducible expression). Even after that, if the protein is not solubilized from the inclusion body then you could try re-solublizing your protein in solubilization buffers and then do dialysis (step-wise if using a denaturant method) to fold your protein. Please read the QIA expressionist handbook for further clarification.
Thank you Roshni Mohan . I will follow the handbook you suggested.
Well, my objective is to purify the protein for studying substrate (phthalate) hydrolysis.
I have tried switching to a pMAL vector (from pET28a vector) to obtain soluble fraction after cell lysis. However couldn't get enzyme activity there, although with pMAL I was able to obtain atleast 20% in soluble fraction (as you can see in the gel image) .
Yes, it is a induced system and the expression host is BL21(DE3).
As you need to perform substarte analysis ensure that your enzyme activity is retained after refolding. Good luck
Here are some general guidelines on solubilizing and refolding inclusion body proteins:
https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/protein-lysis-and-extraction/handling-inclusion-bodies
https://lab.plygenind.com/how-to-solubilize-and-refold-inclusion-bodies-a-practical-guide
Additionally, if solubilization continues to be a problem, you may want to consider strategies to reduce inclusion bodies formation during culture:
https://lab.plygenind.com/how-to-prevent-inclusion-bodies-formation-in-protein-expression
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