pTYB21 is an E. coli cloning and expression vector (7514 bp) used in the IMPACT™ Protein Purification System which allows the overexpression of a target protein as a fusion to a self-cleavable affinity tag (1,2). It is an N-terminal fusion vector designed for in-frame insertion of a target gene into the polylinker, downstream of the intein tag (the Sce VMA intein/chitin binding domain, 55 kDa)(3,4). This allows the N-terminus of the target protein to be fused to the intein tag. The self-cleavage activity of the intein allows the release of the target protein from the chitin-bound intein tag, resulting in a single column purification of the target protein.
This vector can be used in conjuction with a C-terminal fusion vector to test which fusion construction (N-terminal or C-terminal) maximizes the expression and yield of a target protein. For the fusion of the C-terminus of the target protein to the intein tag, use pTXB1 (NEB #N6707), pTXB3 (NEB #N6708), pTYB1 (NEB #N6701), pTYB2 (NEB #N6702), pTYB3 (NEB #N6703) or pTYB4 (NEB #N6704).
- The multiple cloning site (MCS) is compatible with the multiple cloning sites of vectors in the pMAL Protein Fusion and Purification System (NEB #E8200) and the K. lactis Protein Expression Kit (NEB #E1000).
- When the SapI (or BspQI) site in the MCS is used for cloning the 5´ end of the target gene, the N-terminus of the target protein is immediately adjacent to the intein cleavage site. This results in the purification of a target protein without any extra vector-derived residues at its N-terminus. After cloning the target gene in the MCS using SapI, the recognition sequence of SapI is lost; therefore, the vector cannot be recut with SapI. For details, see the IMPACT Manual.
- When NdeI is used for cloning the 5’ end of the target gene, extra amino acids (Gly-Arg-Ala-His) will be added to the N-terminus of the target protein.
- A stop codon should be included in the reverse primer.
- A pBR322 derivative with a ColE1 replication origin.
- Expression of the fusion gene is under the control of the T7/lac promoter and can be induced by IPTG due to the presence of a lacI gene (5).
- Expression requires an E. coli host that carries the T7 RNA Polymerase gene [e.g., T7 Express Competent E. coli, (NEB #C2566) or BL21(DE3) Competent E.coli (NEB #2527) and derivatives].
- Ampicillin resistance.
- When pTYB21 or pTYB22 is used, a small peptide (15 amino acids, 1.6 kDa) is also cleaved from the intein tag and co-eluted with the target protein. It cannot be detected on a regular SDS-PAGE and can be dialyzed out.
- Origin of DNA replication from the bacteriophage M13 allows for the production of single-stranded DNA by helper phage superinfection of cells bearing the plasmid. M13K07 Helper Phage (NEB #N0315) is available.
- Other IMPACT vectors are available which allow for fusion of a target gene to N- or C- terminus of an intein and a cleavage reaction which can be induced by thiol reagent or temperature/pH shift.
- Intein Forward Primer (NEB #S1263) and T7 Terminator Reverse Primer (NEB #S1271) are available for sequencing the target gene.
Properties and Usage
Affinity TagChitin-Binding Domain (CBD)
- Chong, S., Montello, G.E., Zhang, A., Cantor, E.J., Liao, W., Xu, M.-Q., Benner, J. (1998). Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. Nucl. Acids Res. 26, 5109-5115.
- Chong, S., Mersha, F.B., Comb, D.G., Scott, M. E., Landry, D., Vence, L.M., Perler, F.B., Benner, J., Kucera, R.B., Hirvonen, C.A., Pelletier, J.J., Paulus, H., and Xu, M.-Q. (1997). Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene. 192, 277-281.
- Chong, S., Williams, K.S., Wotkowicz, C., and Xu, M.Q. (1998). Modulation of protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein. J. Biol. Chem. 273, 10567-77.
- Watanabe, T., Ito, Y.,Yamada, T., Hashimoto, M., Sekine, S., and Tanaka, H. (1994). The role of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation. J. Bacteriol. 176, 4465-4472.
- Dubendorff, J. W. and Studier, F. W. (1991). Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J. Mol. Biol. 219, 45-49.