Addgene pGEX-2TK
General description
Simplified packaging containing only the pGEX vector in the packaging (E. coli BL21 remains available separately, Code No. 27-1542-01. Updated Product Specification Sheets.
Application
Thirteen pGEX vectors are available (see figure). Nine of the vectors have an expanded multiple cloning site (MCS) containing six restriction sites. The expanded MCS facilitates the unidirectional cloning of cDNA inserts obtained from libraries constructed using many available lambda vectors.
pGEX-6P-1, pGEX-6P-2 and pGEX-6P-3 each encode the recognition sequence for site-specific cleavage by PreScission Protease (see PreScission Protease) between the GST domain and the multiple cloning site. pGEX-4T-1, pGEX-4T-2 and pGEX-4T-3 are derived from pGEX-2T and contain a thrombin recognition site. pGEX-5X-1, pGEX-5X-2 and pGEX5X-3 are derivatives of pGEX-3X and possess a Factor Xa recognition site.
pGEX-2TK is designed to allow the detection of expressed proteins by directly labeling the fusion products in vitro (1). This vector contains the recognition sequence for the catalytic subunit of cAMP-dependent protein kinase obtained from myocardium. The protein kinase site is located between the GST domain and the MCS.
Expressed proteins can be directly labeled using protein kinase and [gamma-32P]ATP and easily detected using standard radiometric or autoradiographic techniques. pGEX-2TK is a derivative of pGEX-2T; the fusion proteins can be cleaved with thrombin.
Cleavage of pGEX-6P GST fusion proteins occurs between the Gln and Gly residues of the recognition sequence Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro (2). Low temperature (5°C) digestion minimizes the degradation of the protein in question.
Since PreScission Protease is designed with a GST tag, it can also be removed from the splice mixture simultaneously with the GST portion of the fusion protein.
The pGEX-6P expression vectors allow for easy site-specific cleavage and simultaneous purification on glutathione-sepharose. The pGEX-6P series provides all three translational reading frames linked between the GST coding region and the multiple cloning site.
Features and benefits
- A tac promoter for chemically inducible high-level expression of GST-tagged recombinant proteins.
- An internal lacIq gene for use in any E. coli host.
- Very mild elution conditions for release of fusion proteins from the affinity matrix, minimizing the effects on antigenicity and functional activity.
- PreScission™ recognition sites for protease, thrombin or factor Xa to cleave the desired protein from the fusion product.
Analysis Note:
To view the Certificate of Analysis for this product, visit www.cytiva.com.
Legal information
pGEX vectors
pGEX vectors may be used for scientific research and investigation and for no other purpose and a license for commercial use of the licensed products and processes claimed in U.S. Patent 5,654,176 and equivalent patents and patent applications in other countries must be obtained directly. Negotiated with Millipore Corp (formerly Chemicon International Inc) by purchaser prior to such use.