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Baculovirus system -swap tag

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#1 AK123



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Posted 19 February 2009 - 02:00 PM


I am currently using the pAcSecG2T vector (BD BioSciences). This is a transfer vector containing gp67 signal sequence at the N-terminus, followed by GST-tag, then the protein of interest. My previous attempt to express a protein using this system was successful. However, for some reason, the GST-fusion protein does NOT bind to my glutathione column. I suspected that the GST tag is somehow affecting the proper folding/conformation of my protein. Therefore, I tried to replace the GST tag with a 10xHis tag. Now, using this newly constructed transfer vector (sequence verified), I did the transfection again. However now, I could not detect the production of His tagged protein. I have attempted the transfection twice... still no protein detected in tranfection (media collected 4 days post transfection) by western with anti-His antibody.

I am wondering if anyone has attempted to do this & has success. I can't think of any reason why this should not work...

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