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I noticed that there are 116 additional residues before the His-tag in the N-terminal and 38 additional residues after the His-tag and before start of the protein sequence. I would like to know the functionality and rationality behind attaching these constructs. I would like to use the protein in a functional assay and would also like to know if you have any data regarding binding affinity, turnover rate, and such.
Asked on Nov 20 2014
HIF1alpha is a large protein to express recombinantly in E.coli so a few elements were added to aid in expression/solubility/purification. This construct has an N-terminal Trx – His Tag followed by a linker and a TEV cleavage site.
-Trx (thioredoxin) added to aid solubility.
-HIS for affinity purification
-TEV cleavage site (ENLYFQG) added as a way to remove the tag from the HIF sequence by adding highly purified TEV protease.
We know this protein is recognized by anti-HIF antibodies on western blot and is functional in a sandwich ELISA, however we do not have any functional data for this construct.
Jeremy Kasanov Abcam Scientific Support
Answered on Nov 20 2014