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The Use of TAGZyme for the Efficient Removal
of N-Terminal His-Tags
José Arnau, Conni Lauritzen, Gitte Ebert Petersen, and John Pedersen
Summary
The use of affinity tags and especially histidine tags (His-tags) has become widespread
in molecular biology for the efficient purification of recombinant proteins. In some cases,
the presence of the affinity tag in the recombinant protein is unwanted or may represent a
disadvantage for the projected use of the protein, like in clinical, functional or structural
studies. For N-terminal tags, the TAGZyme system represents an ideal approach for fast
and accurate tag removal. TAGZyme is based on engineered aminopeptidases. Using
human tumor necrosis factor as a model protein, we describe here the steps involved in
the removal of a His-tag using TAGZyme. The tag used (UZ-HT15) has been optimized
for expression in Escherichia coli and for TAGZyme efficiency. The UZ-HT15 tag and
the method can be applied to virtually any protein. A description of the cloning strategy
for the design of the genetic construction, two alternative approaches and a simple test to
assess the performance of the tag removal process are also included.
Key Words: Histidine tags; N-terminal tag; affinity tag removal; aminopeptidases;
TAGZyme; downstream processing; recombinant protein.
1. Introduction
Affinity chromatography has become the method of choice to simplify
and improve recovery in the purification of recombinant proteins. Affinity
chromatography currently represents the most powerful tool available to
From: Methods in Molecular Biology, vol. 421: Affinity Chromatography: Methods and Protocols, Second Edition
Edited by: M. Zachariou © Humana Press, Totowa, NJ
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