TAGZyme System

Yes. In order to cleave off an N-terminal 6xHis tag, a protease cleavage site must be inserted between the coding sequences of the tag and the N-terminus of the protein. Factor Xa Protease recognizes the amino acid sequence Ile-Glu-Gly-Arg and cleaves the peptide bond C-terminal of the arginine residue. The expression vector pQE-30 Xa encodes a Factor Xa Protease recognition site between the N-terminal 6xHis-tag sequence and the multiple cloning site.

If the gene of interest is cloned blunt ended at the 5´-end using the StuI restriction site of the vector, Factor Xa Protease cleavage of the purified recombinant protein results in a protein product without any vector-derived amino acids at the N-terminus.

Tags can also be removed exoproteolytically using the TAGZyme System. This system is an efficient and specific solution for the complete removal of small N-terminal His tags and other amino acid tags by the use of exopeptidases. For detailed information on the procedure please review the TAGZyme handbook.

Please note that both tag removal options work on N-terminal 6xHis tags only.

 

 

DAPase (dipeptidyl aminopeptidase I = Cathepsin C), even though endogenously expressed in human tissues and cells, will have only a negligible effect on the degradation of proteins expressed in cell culture. The reason for this is the low level of endogenous DAPase compared to the much higher level of typically overexpressed recombinant protein. However, it is always worthwhile to run a time-course expression experiment to check for possible protein degradation, since proteases and peptidases tend to degrade proteins over time.

The removal of DAPase is >99%.

DAPase: heterodimer, 24 kDa and 6 kDa subunit; Qcyclase: 35 kDa; pGAPase: 28 kDa

Yes. The TAGzyme enzymes do not experience any loss in function after several freeze and thaw cycles. However, storage at -80°C is not necessary as storage at -20°C is adequate.