Cell Free Expression Application: Production of Soluble Protein for Structural Analysis

The TNT® SP6 High-Yield Protein Expression System uses a high-yield wheat germ extract supplemented with SP6 RNA polymerase and other components. Coupling transcriptional and translational activities eliminates the inconvenience of separate in vitro transcription and purification steps for the mRNA, while maintaining the high levels of protein expression. All that is required is the addition of DNA templates containing the SP6 promoter and the protein coding region for the protein of interest. Furthermore no specialized equipment is required for protein screening and production. The system enables the expression of approximately 100µg/ml of protein in batch reaction and 200–440µg/ml in dialysis reaction in 10–20 hours .

In a recent publication (Zhao, L. et.al. (2010) J. Struct. Genomics 11, 201–9), the Northeast Structural Genomics Consortium (www.nesg.org) in their quest to express 5,000 eukaryotic proteins, report that even with different cloning strategies they could only produce 26% of the proteins in a soluble form. To improve the efficiency of expressing soluble protein, they investigated the use of wheat germ cell free system as a alternative to E.coli.

In this publication 59 human constructs were expressed in both E.coli and the wheat germ cell free system. Only 30% of human proteins could be produced in a soluble form using E.coli -based expression. Some 70% could be produced using the TNT® SP6 High Yield Wheat Germ system.
To further demonstrate the utility of expressing proteins that were suitable for structural studies from a wheat germ-based system, two of the proteins were isotope enriched and analyzed successfully by 2D NMR.