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GST fusion protein - two dominant bands

GST Recombinant Purification

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3 replies to this topic

#1 Missle

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Posted 10 May 2016 - 12:12 PM

Hello.  I'm new to GST purification.  I expressed a fusion protein: GST-'Protein X'.  I can see expression in the soluble lysate upon over-expression.  When purified with Glutathione Sepharose, the A280 reading of the eluent would indicate a ~0.5-0.8 mg/mL concentration yet when loading the sample, I'm not getting anywhere near that amount of protein.  I blanked with elution buffer.

 

Additionally, the SDS-PAGE shows a faint band corresponding to the fusion protein and a predominant band around the size of GST alone (which is the same size as 'Protein X').  Does anyone know how I could get GST alone?  There is a thrombin cleavage site but I didn't perform the cleavage!  Any ideas would be much appreciated!



#2 labtastic

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Posted 10 May 2016 - 04:35 PM

Do you have triton or anything in your buffer that would absorb at 280nm instead of protein? Or doe the spectrum look more like DNA (max at 260nm)?

 

Sounds like you've got some proteolytic cleavage happening from endogenous ecoli proteases. This is not as unusual as you would hope.

 

The two simplest solutions are (i) use protease inhibitors when you lyse your cells or (ii) use shorter inductions times (not sure how long your expressing for but sometimes shortening this time can help).

 

If those don't work, consider a different fusion protein (SUMO, GFP).

 

Also consider Prescission Protease site instead of thrombin. This is advantageous because you once you cleave the GST from your protein, you can re-run the mix over the GST column and remove all cleaved GST from your protein as well as the prescission protease. You can't do this with thrombin since it does not come with affinity tags.



#3 Missle

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Posted 12 May 2016 - 08:21 AM

Thank you labtastic.  After I posted, I was wondering about premature translation termination.  The target protein does contain some repeat sequence at the N-term.  Thoughts?



#4 labtastic

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Posted 25 May 2016 - 03:45 PM

I have heard lots of people talk about pre-mature translation termination but I have yet to run across this as a serious issue in my experience. 

 

Nevertheless, if this is the problem, you can solve it by putting in a c-terminal his tag. This way you only purify full-length protein and not GST alone (whether the free GST is caused by proteolysis or pre-mature truncation). Then bind your protein to a GST column if you need and continue as usual.







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