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gst-fusion protein, mainly


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#1 ozgurm

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Posted 07 October 2004 - 01:23 AM

Hi All,
I have to make a decision, I think I need your help with this. I am tyring to get soluble forms of a 30 kD protein expressed. I have used pet17b and pgex4t-1 vectors, expression conditions were 0.4 M IPTG induction, 37 degrees centrigrate, 0,1,2,3,4 hours sampling. FROMthe results, I got various kinds of bands in both sds page and western blots. I could not determine whether my protein is expressed or not but according to the molecular weight of my protein, it is expressed and found in the nonsoluble part as well as fairly seen in soluble part. I have tried one more different condition, 20 degrees and o.1 M IPTG induction but yielded the same results for pgex4t-1 ( did not try for pet17b).
Do you have any idea to get this protein in soluble form and get distinct, clear band appearrance? I have used rabbit to immunize it with a 7aa peptide to get antisera.
Thank you

#2 senmehmet

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Posted 10 October 2004 - 08:05 PM

Hello
1-Try 15celcius expression but worked for me last week
2-Try to purify cell lysate by GST coloumn., if you can find anything in the cell-lysate as soluble, it is yours. (i hope you aren't working NMR where you need mM protein, I know b/c I am doing NMR)
3- If your protein is going to insoluble fraction, try to refold it and purify it again.
Good Luck
Mehmet

#3 Dizz

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Posted 12 October 2004 - 01:13 PM

First, for the clear band problem, it's possible that there is partial degradation during the expression and/or purification steps but normally it is not a big deal. Also, always use un-induced control in your gel when you analyse the expression of the protein. Normally, you don't have to do western blot to see you're recombinant protein.

For the soluble problem it is more difficult. If there is no protein in the soluble fraction even at 20 or 15 degrees you would have to denature in urea and renature using dialysis.

You could also try to add glycylglycine (500mM-1000mM) in the culture media (LB or TB), it as been shown to increase the solubility.




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