removal of his tag - (Jan/25/2007 )

hi all!
can anyone give me any tips on removal of my his tag from pET30 Ek/lic? normally thrombin cleavage is used?yes/no? i dont have that much grant money to be buying expensive kits and i would only have to do a small scale cleavage. any ideas?
thanks in advance

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I know GST-tags to cleave by thrombin beside hexahistidinyl is combined with a thrombin cleaving site (I do not know your vector - you may know if there is a 6His with thrombin cleavage site);

once, 6His-tag was launched to make cleavage superfluous as in many cases 6His may not disturb the function of the expressed protein; think, if cleaving is necessary...

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if your fusion protein contains enterokinase active site then you can use the enterokinase enzyme for your Hig tag removal from the protein.first check in your plasmid construct.

enterokinase :
Description:
Enterokinase is a specific protease that cleaves after lysine at its cleavage site Asp-Asp-Asp-Asp-Lys.
It will sometimes cleave at other basic residues, depending on the conformation of the protein substrate. Enterokinase will not cleave at site followed by proline.

Specificity:
Enterokinase is a specific protease that cleaves after lysine at its cleavage site Asp-Asp-Asp-Asp-Lys'.

Molecular Weight:
Theoretical: 26.3 kDa and Apparent: 31 kDa

Cleavage of fusion proteins at definite cleavage sites. This recognition sequence, however, can also be
used as a restriction cleavage site for processing recombinant proteins (3). For this purpose, the desired protein is fused at the C-terminal of the recognition sequence. After purification of the entire fusion protein, the protein or peptide is released by incubation with enterokinase. The release of the desired protein or peptide
component from a fusion protein is affected by
the adjacent amino acid sequences at the cleavage site as well as by the size of the two fused components and by the accessibility of active site.

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hi. thanks for the reply! im wanting to run the cleaved and uncleaved protein on CD spectra to prove to my collaborator the tag has no effect. there is a thrombin cleavge site on it and it is 6his. how do i cleave it though?

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