Enzymes: biotin ligase from E. coli (EC 188.8.131.52); protein disulfide isomerase (EC 5.3.41).
Efficient synthesis of a disulfide-containing protein through a batch cell-free system from wheat germ
Article first published online: 28 NOV 2003
European Journal of Biochemistry
Volume 270, Issue 23, pages 4780–4786, December 2003
How to Cite
Kawasaki, T., Gouda, M. D., Sawasaki, T., Takai, K. and Endo, Y. (2003), Efficient synthesis of a disulfide-containing protein through a batch cell-free system from wheat germ. European Journal of Biochemistry, 270: 4780–4786. doi: 10.1046/j.1432-1033.2003.03880.x
- Issue published online: 28 NOV 2003
- Article first published online: 28 NOV 2003
- (Received 4 August 2003, revised 30 September 2003, accepted 20 October 2003)
- cell-free synthesis;
- wheat germ;
We have developed a highly productive cell-free protein synthesis system from wheat germ, which is expected to become an important tool for postgenomic research. However, this system has not been optimized for the synthesis of disulfide-containing proteins. Thus, we searched here for translation conditions under which a model protein, a single-chain antibody variable fragment (scFv), could be synthesized into its active form. Before the start of translation, the reducing agent dithiothreitol, which normally is added to the wheat germ extract but which inhibits disulfide formation during translation, was removed by gel filtration. When the scFv mRNA was incubated with this dithiothreitol-deficient extract, more than half of the synthesized polypeptide was recovered in the soluble fraction. By addition of protein disulfide isomerase in the translation solution, the solubility of the product was further improved, and nearly half of the soluble polypeptides strongly bound to the antigen immobilized on an agarose support. This strong binding component had a high affinity as shown by surface-plasmon resonance analysis. These results show that the wheat germ cell-free system can produce a functional scFv with a simple change of the reaction ingredients. We also discuss protein folding in this system and suggest that the disulfide bridges are formed cotranslationally. Finally, we show that biotinylated scFv could be synthesized in similar fashion and immobilized on a solid surface to which streptavidin is bound. SPR measurements for detection of antigens were also possible with the use of this immobilized surface.