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how to isolate insoluble protein in bacterial induction?


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#1 leomap

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Posted 10 September 2009 - 07:19 AM

Hi,

I am trying to expressed a piece of my protein of interest in PGEXT4T1 vector (GST tag). I induced the cell at OD 0.6 with IPTG for 3hrs at 30C. However, the protein is insoluble and stayed in the pellet after sonication. I tried to treat the pellet with triton X-100 and load the whole complex and probe with GST and see the protein (a band with the corresponded size). I read in other topics people talking about inducing at lower temperature for a longer period of time. I just wonder if there are other alternatives to move the proteins out of the pellet?

Thanks
Leo

#2 swanny

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Posted 10 September 2009 - 04:02 PM

Some people have thought about using autoinduction media to slowly switch on the transcription machinery. Whether this works in your case (or any specific case, let's be fair) will only be found by doing the experiment.

Failing that, temperature and IPTG concentration are the standard ways to try to reduce protein going into the insoluble fraction.

As a complete alternative, will your project suffer if you let the protein go insoluble and purify by denaturing it and refolding? Some inclusion bodies have been teased apart by slowly increasing the urea concentration, kind of like an ammonium sulphate precipitation in reverse. St out at 1 M, which is often used to clean other contaminating proteins off the surface of the IB. You'll have to go slowly, adding the urea, incubating, spinning down to see what happens to the pellet and adding more urea as appropriate. Real old-school, bootstrap stuff, but it might work if you don't want to smash the IB into submission with 8M urea.
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#3 paramyosin

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Posted 14 September 2009 - 08:21 AM

If you want to purify your fusion protein by binding GST, you can not use as much urea as 8 M, but with 1.5-2 M urea and maybe 0.1% emulphogen or triton you can obtain some solubilization from the insoluble fraction and can still be able to bind GST.

Good Luck
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#4 Doc Sheldon

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Posted 14 September 2009 - 02:34 PM

I agree with the comments already posted. The basic idea is to slow down the expression so the protein has time to fold correctly. There are some other tricks you can try and my team has seen lots of similar cases to yours. The two documents I uploaded for you outline the approaches we use for solving the solubility challenge. (Forgive the commercial aspect of the papers, but they do have some excellent references you will find helpful.) Short of finding a means of producing soluble protein, we purify the inclusion bodies, denature and refold. Brutish but workable. Let me know if you need any more advise.

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#5 Yar

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Posted 05 October 2009 - 07:17 AM

You can also try expressing with GroES/EL. Also if you want to unfold you can put a His tag on your protein and purify in denaturing conditions (8M Urea). The His tag will still bind Nickle when denatured. Just my two cents.




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