Wheat germ cell-free protein expression system
The translational apparatus in Escherichia coli (E.coli) is an important tool in the expression and structure of proteins. However, restricted expression due to the presence of a cell membrane leads to comparably less yield than the wheat germ cell-free protein expression tool. The wheat germ cell-free protein expression tool creates an optimal environment for protein synthesis due to all metabolic resources being assigned to the protein in question as opposed to basic cellular mechanisms.
Overexpression of proteins in cell-dependent systems can lead to misfolded proteins with low solubility. The eukaryotic cell-free wheat germ protein expression system provides the cellular machinery for the production of proteins with complex conformational shapes, such as disulfide-bond-containing or integral membrane proteins.
The mRNA translated has a 5’ cap and a poly(A) tail, which in combination with the highly stable and specific wheat germ extract, is translated into protein. As agents that aid protein folding can directly be added to the reaction, this allows the delivery of protein with a correct three-dimensional structure, essential for their biological function. Translation is carried out in combination with a liposome permitting the specific protein structures to be maintained in the purification stage. Consistency in production is achieved using robotic methods and studies have shown that this wheat germ cell-free system is an effective method in expressing complex protein shapes with high solubility.
In vitro transcription is carried out using a DNA template in form of a plasmid. The DNA is transcribed in vitro using a transcription kit. Wheat germ extract containing all the necessary requirements for translation is added to the reaction and the mRNA is then translated into protein. The newly transcribed proteins are transferred into liposomes, which allow native protein shapes to be maintained during the washing stages.
Advantages of wheat germ expressed proteins
- Animal- and serum-free
- Of eukaryotic origin and therefore complexity in shape maintained
- High solubility due to native conformation and eukaryotic source
- Greater stability
Categories included are: cluster of differentiation (CD) Molecules, G protein coupled receptors (GPCR), ion channels, transporters
View a selective range of wheat expressed proteins:
Vinarov, A.V.; Loushin Newman, C.L and Markley, J.L. (2006) FEBS Journal 273, 4160-4169
Guild, K.; Zhang, Y; Stacy, R.; Mundt, Benbow, S.; Green, A.and Myler P.J (2011) Acta Crystallogr Sect F Struct Biol Cryst Commun. 97(Pt 9), 1027-1031
Goren, M.A; Nozawa, A.; Makino, S; Wrobel, R.L.; Fox, B.G. (2009) Methods Enzymol. 463, 647-673