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Silly question on His-tagged protein - (Oct/07/2005 )

Hi. I need your advices for my silly question

My protein gets inactive when it has N-terminal His tag.

And my recombinant plasmid has N-terminal His tag and following thrombin siten in the upstream of the CDS.

Can I just cut the His tag by thrombin to get the active form?

Thanks in advance.

-yja97-

It depends if the tag has reversible altered the conformation of the protein. Try it.

QUOTE (yja97 @ Oct 7 2005, 10:11 AM)
Hi. I need your advices for my silly question

My protein gets inactive when it has N-terminal His tag.

And my recombinant plasmid has N-terminal His tag and following thrombin siten in the upstream of the CDS.

Can I just cut the His tag by thrombin to get the active form?

Thanks in advance.

-Moz-

If your plasmid looks like this, it can be cleaved.
Start (N-terminal) – target protein – C-terminal - Thrombin – His Tag
It can be cleaved by thrombin and remove the His fusion tag.

Please note that the protease cleavage site should be in between your target protein and the His tag.

-sallylyc-