protein expression (temperature issue) - trying to express soluble protein at lower temperature (Oct/25/2009 )

Hi,

I am trying to express a protein from pGEX-5X-1 vector. It was found to be in the inclusion body when expressed at 37C and 30C. I would like to express the protein in the soluble form by lowering the temperature. I am thinking of trying the expression at 18C.

I see that usually papers would induce around 18 hours. I would like to know if 5 hours would be enough to produce the protein?
Any suggestions?

Thanks in advance.

At 18 degrees, an overnight expression would probably be better.

If you're still not having any joy, some people have started using autoinduction media for proteins with lower solubility, because the autoinduction process appears to be gentler than the IPGT induction sledgehammer.

Hi,
I think you can try induction at 18 degrees.
But I would suggest you take hourly samples may be for first 6 hours and then finally after 18 hours.
Check all the samples on SDS PAGE and then decide how much time you should incubate..

regards,
Kaushik

simlez on Oct 25 2009, 09:25 PM said:

a more complicated solution can be adition of a soluble fusion tag!!!
as kaushik says you can do a qualitative test by SDS or if you are also interested in the yield or expression, try developing an HPLC method!!!
Best luck!!!

Pradeep Iyer on Oct 26 2009, 04:44 PM said:

DRN on Oct 27 2009, 06:12 PM said:

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Thanks all for the replies!

I've tried O/N at 18C... It seems that the protein is still expressed mainly as inclusion bodies.

I also tried 5 hours at 18C. It is enough to express the protein but the yield is much lower and the protein is still expressed as inclusion bodies.

Also, I have another problem. My target protein seems to be very close to host cell protein when I run sds page for my uninduced and induced sample. I have no clue whether is it leaky expression or is it host cell protein.

Any suggestion on how to differentiate them?

Thanks in advance!

simlez on Oct 31 2009, 06:32 AM said:

That's the problem.. I think my lab doesn't have western...

is there any other method besides western?