TnT® Coupled Wheat Germ Extract System

The TnT® Coupled Wheat Germ Extract Systems offer an alternative for eukaryotic cell-free protein expression: a one-tube, coupled transcription/translation system. The TnT® Extract Systems greatly simplify the process and reduce the time required to obtain in vitro translation results. Standard wheat germ extract translations commonly use RNA synthesized in vitro from SP6 or T7 RNA polymerase promoters. This entire process requires separate reactions with several steps between each reaction. The TnT® Extracts bypass many of these steps by incorporating transcription directly in the translation mix. Additionally, the TnT® Extract reactions often produce significantly more protein (two- to sixfold) in a 1.5-hour reaction than do standard in vitro wheat germ extract translations using RNA templates.

Magnesium Acetate, 25mM, and Potassium Chloride, 2.5M, can be used to optimize in vitro translation reactions in the TnT® T7 Quick Coupled Transcription/Translation System, Flexi® Rabbit Reticulocyte Lysate System and TnT® Coupled Wheat Germ Extract System.

To produce protein from linear plasmids, choose the T7 promoter. The SP6 promoter will produce protein from circular DNA. For PCR templates use TnT® T7 Quick for PCR DNA (Cat.# L5540). Luciferase Control DNA and Luciferase Assay Reagents are included with the system as functional controls. Only full-length luciferase is active.

For more information, see the Protein Expression chapter of the Protocols & Applications Guide.

Applications

• Protein:protein interactions.
• Protein activity studies.
• The Wheat Germ Extract System is particularly suited to translation of mammalian transcription factors, since it does not contain mammalian transcription factors.
• Protein:nucleic acid interactions.
• Simultaneously express multiple genes that are under the control of different phage polymerase promoters.
• Rapid screening and analysis of mutants.
• Protein Truncation Test (PTT).

References

1. Anderson, C.W. et al. (1983) Meth. Enzymol. 101, 635–44.
2. Krieg, P.A. and Melton, D.A. (1984) Nucl. Acids Res. 12, 7057–70.