Affinity tags as fusions to the N- or C-terminal part of proteins are valuable tools to facilitate the production and purification of proteins. In many cases, there may be the necessity to remove the tag after protein preparation to regain activity. Removal of the tag is accomplished by insertion of a unique amino acid sequence that is recognized and cleaved by a site specific protease. Here, we report the construction of an expression vector set that combines N- or C-terminal fusion to either a hexahistidine tag or Streptag with the possibility of tag removal by factor Xa or recombinant tobacco etch virus protease (rTEV): respectively. The vector set offers the option to produce different variants of the protein of interest by cloning the corresponding gene into four different Escherichia coli expression vectors. Either immobilized metal affinity chromatography or streptactin affinity chromatogaphy can be used for the one-step purification. Furthermore, we show the successful application of the expression vector for C-terminal hexahistidine tagging. The expression and purification of His-tagged L-2-hydroxyisocaproate dehydrogenase yields fully active enzyme. The tag removal is here accomplished by a derivative of rTEV. (C) 2004 Elsevier Inc. All rights reserved.