pet-32 - (Jan/05/2015 )

Hi all.

According to the fact that pet-32 vector contains two his-tag (internal and terminal), how can I remove the thioredoxin tag after cleavage with enterokinase ? (After cleavage, one his-tag remains attached to my peptide, and another his-tag to the thioredoxin and i have not designed any additional cleavage site).  

if the proteins are sufficiently different in size then you can use gel filtration.

you may also try ion exchange.

Use a stop codon before the C-terminal his-tag.

When you clone your gene into the vector, use a reverse primer that encodes the c-terminal end of your protein of interest, followed by a stop codon, then the appropriate restriction site (BamHI, EcoRI, HindIII, NotI, XhoI). Expression will then stop before it reaches the C-terminal His-tag of the vector.

Cleave with enterokinase, run the mixture through a nickle column and the thioredoxin will bind, while your protein will flow thru.

Thanks for your helps