General descriptionThe pET-41a-c(+) and pET-42a-c(+) vectors incorporate the schistosomal glutathione-S-transferase (GST; GST•Tag) coding sequence as a fusion partner. The GST•Tag sequence has been reported to enhance the production and in some cases the solubility of its fusion partners (Smith 1998). When expressed in a soluble, properly folded form, GST•Tag fusion proteins can be purified with immobilized glutathione. Gentle elution is achieved with buffers containing reduced glutathione. Quantification of soluble GST fusions is also possible by assaying the transferase activity. The pET-41 and -42 series feature the powerful T7lac promoter, and encode the GST•Tag (220 aa) sequence, proteolytic sites, His•Tag (6 aa) sequence, and S•Tag (15 aa) sequence.
In contrast to other commercially available GST-fusion vectors, the Xa (IleGluGlyArg, pET-42 series) and enterokinase (AspAspAspAspLys, pET-41 series) proteolytic cleavage sites have been engineered to allow removal of 100% of the vector-encoded sequences and the generation of native proteins with their authentic N-terminal residues. Unfused proteins can be produced by using the Nde I cloning site. A version of pET-41 is available as a linearized vector prepared for ligation-independent cloning (LIC) of PCR products.
Another pET vector with the GST•Tag sequence is pET-49b(+). Please see the website description for more information. The His•Tag and S•Tag sequences enable alternative protein detection and purification procedures to be performed. For example, when enhancing purity with a separate purification method, or when purifying under denaturing conditions.
The pET-41 series is designed for cloning and high-level expression of peptide sequences fused with the 220 aa GST•Tag protein. Note that the sequence is numbered by the pBR322 convention, so the T7 expression region is reversed on the vector map (TB239). The f1 origin is oriented so that infection with helper phage will produce virions containing single stranded DNA that corresponds to the coding strand. Vector encoded sequence can be completely removed when cloning into the PshAI site (as shown below) and then cleaving the GST fusion protein with Enterokinase.
The pET Vectors are supplied as purified plasmid DNA (10 µg). Each order of pET DNA also includes an Induction Control strain (supplied as a glycerol stock). Please contact technical service if you need additional information.
Other NotesSmith, D.B. and Johnson, K.S. 1988. Gene 67, 31.
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