What vectors are included in the IMPACT-CN kit?
The IMPACT-CN kit includes four E. coli expression vectors (pTYB1, pTYB2, pTYB11, pTYB12) which allows for the fusion of the cleavable intein tag to either the C-terminus or N-terminus of the target protein. The IMPACT-CN System utilizes an intein from the Saccharomyces cerevisiaeVMA1gene [Kane, et al., (1990) Science,250, 651-657]. The C-terminal fusion vectors, pTYB1 and pTYB2, use the 454-residue Sce VMA intein, which has been modified to undergo a self-cleavage reaction at its N-terminal junction at 4°C when induced by thiol reagents such as DTT. pTYB1 and pTYB2 use ATG of the NdeI site in the multiple cloning region for translation initiation. The gene encoding the target protein is inserted into the multiple cloning site to create a fusion between the C-terminus of the target gene and the N-terminus of the gene encoding the intein. DNA encoding a chitin binding domain (CBD) has been added to the C-terminus of the intein to allow affinity purification of the 3-part fusion. pTYB11
