Cell-free protein synthesis has emerged as powerful alternative to cell based protein expression for functional and structural proteomics. The TNT® SP6 High-Yield Protein Expression System uses a high-yield wheat germ extract supplemented with SP6 RNA polymerase and other components. Coupling transcriptionaland translational activities eliminates the inconvenience of separate in vitro transcription and purification steps for the mRNA, while maintaining the high levels of protein expression (1). 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.
Using this system in a dialysis reaction, we achieved protein expression levels that allow visualization of expressed proteins on Coomassie® blue stained SDS PAGE Gels. 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 (1).
In addition to high yields, the wheat germ-based system expressed more full-length, soluble proteins when compared to cell based E.coli expression. Only 3 of the 55 human proteins evaluated were expressed in the soluble fraction using E.coli, while all 55 were expressed as full-length and soluble in the wheat germ system (2).
With these levels of expression, enzyme activities can be assayed directly in the extract without further purification. Simple purification can be achieved with the addition of an affinity tag to the expressed protein. Efficient protein labeling as analyzed by MALDI-TOF was achieved using seleno-methionine and [U-14N] with incorporation of >95% using a modified extract in which the corresponding unlabeled amino acids were substituted by labeling alternatives.
For additional information:
- Zhao, K-Q. et al. (2007) J. Funct. Genomics. 8,199–208
- Slater, M. et al. (2005) Promega Notes. 91, 21–25
Latest posts by Gary Kobs (see all)
- HaloTag Application: Fluorescence Under Stress - May 11, 2018
- Beer Is Complicated: Proteome Analysis via Mass Spectrometry - April 11, 2018
- Characterizing Multi-Subunit Protein Complexes Using Cell-Free Expression - March 12, 2018