Abstract
The TAGZyme system allows efficient and precise exoproteolytic cleavage of N-terminal affinity tags, such as the 6xHis tag, from recombinant proteins. In combination with Ni-NTA technology, the TAGZyme system provides high-purity proteins free of vector-encoded amino acids for use in applications that demand recombinant reagents, an absence of nonspecific cleavage, and a complete removal of all impurities from the target protein preparation. We present results of recent studies on the use of the TAGZyme system. The N-terminal affinity tag sequences encoded by the TAGZyme pQE expression vectors are optimized with respect to their cleavage using TAGZyme exoproteases. The vectors have multiple cloning site sequences designed to allow complete exoproteolytic removal of the encoded N-terminal affinity tag regardless of the restriction site used for cloning. The efficient cleavage reaction and removal of the TAGZyme enzymes by subtractive Ni-NTA chromatography is demonstrated for 6xHis-interleukin-1beta and 6xHis-TNF. In both cases, more than 99.8% of TAGZyme proteolytic activity was separated from recovered, detagged proteins. N-terminal analyses by Edman degradation revealed the predicted sequences of the native proteins and indicated a purity in excess of 99%. For cleavage of both 6xHis-tagged GFP and IL-1beta, DAPase enzyme gave an average cleavage rate of 1.5 +/- 0.5 min per amino acid.