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pGEX vectors and GST expression

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3 replies to this topic

#1 BSturt



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Posted 21 December 2010 - 07:51 AM

Can I use any of the pGEX vectors to express GST alone? I don't want to clone anything into the vector to get a GST tagged protein, I just want GST alone.

I'm using a antibody purification protocol that calls for me to incubate serum with antigen (GST fusion) and GST protein.

Thanks for your help.

#2 protolder



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Posted 21 December 2010 - 11:09 PM

Hola, if you donīt fuse anything to gst the system only will give GST. Moreover sometimes inducing GST-tagged proteins a portion of GST alone appears. Buena suerte

#3 MyProteinBulliedMe



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Posted 22 December 2010 - 02:30 AM

That happened to me too, I tried to express GST-tagged and I got GST alone and my GST-tagged protein in somewhat equal amount.

Edited by MyProteinBulliedMe, 22 December 2010 - 02:30 AM.

#4 paramyosin



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Posted 29 December 2010 - 07:21 AM

Yes, you just have to use the pGEX plasmid and express it without cloning anything downstream, in the MCS, but take into account that maybe you will have in the C-terminal of your GST, the target sequence of a protease or another tag, depending on the plasmid. if you want to avoid those you could amplify the GST gen from this plasmid with a stop codon in your primer at 3ī and clone it in another plasmid.
Regarding the other comentary, sometimes this kind of plasmids have a protease target between GST gene and the MCS. For example, I have noticed in the case of factor Xa, i am convinced that there is a specific degradation of the recombinant protein when the complete protein is been producing due to traces of bovine factor Xa (maybe ppm) which are contaminating the media and the lysed extract also. Due to this reason we can have GST alone and GST plus the target protein.
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