pTYB22 Vector

Features

• 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).
• After the cleavage of the intein tag, a target protein is obtained with extra residue(s) added to N-terminus. For instance, cloning the 5´end of a target gene using NdeI site in pTYB22 adds extra three residues (Ala-Gly-His) to the N-terminus of the target protein. 
• A stop codon should be included in the reverse primer.
• Compatible sites are present in both pTYB22 (eg. NdeI and EcoRV) and pTYB2 (eg. NdeI and SmaI; Other IMPACT vectors are available which allow for fusion of a target gene to N- or C- terminus of an intein. The cleavage reaction can be induced by thiol reagent or temperature/pH shift.
• 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 (High Efficiency),  (NEB #C2566)  or BL21(DE3) Competent E. coli (NEB #C2527) 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.

Companion Products

• BL21(DE3) Competent E. coli
• T7 Express Competent E. coli (High Efficiency)

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.

• InteinMediated Protein Ligation IPL and Labeling with the IMPACT Kit N6710

• IMPACT™ Vectors and Applications

• DNA Sequences and Maps Tool

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The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

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New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

Licenses

 

The buyer and user have a non-exclusive sub-license to use this system for any component thereof for RESEARCH PURPOSES ONLY, based upon agreement to the following assurances.

Transfer of this vector in host cells that contain the cloned copy of the T7 gene 1 to third parties is explicitly prohibited.