描述

pTYB4 is an E. coli expression vector (7,474 bp) used in the IMPACT™ Kit (NEB #E6901) (1). This C-terminal fusion vector is designed for the in-frame insertion of a target gene into a polylinker upstream of an intein tag (the Sce VMA intein/chitin binding domain, 55 kDa) (1,2). This results in the fusion of the C-terminus of the target protein to the N-terminus of the intein tag. Thiol-induced self-cleavage of the intein releases the target protein from the chitin-bound intein tag, resulting in a single column purification of the target protein (3).

特色

• The NcoI site in the polylinker contains an ATG sequence for translation initiation.
• Use of the SmaI site for cloning will yield a target protein with a single glycine residue added to its C-terminus after cleavage.
• Unique sites indicated in bold.
• Expression of the fusion gene is under the control of an IPTG-inducible T7 promoter (4).
• Origin of DNA replication from bacteriophage M13, which allows for the production of single-stranded DNA by helper phage superinfection of cells bearing the plasmid (M13K07 Helper Phage, NEB #N0315)
• A pBR322 derivative with a ColE1 derivative origin
• Ampicillin resistance.
• Other IMPACT vectors are available which allow for fusion of a target gene to N-or C- terminus of an intein. The cleavage reaction may be induced by thiol reagent or temperature/pH shift.
• Companion vectors (pTYB1, pTYB2, pTYB3) differ only in the sites present in the polylinker.
• A wide range of E. coli host strains: T7 Express Competent E. coli (High Efficiency) (NEB #C2566) or BL21(DE3) Competent E. coli (NEB #C2527); and derivatives.