tailieunhanh - Báo cáo khoa học: Efficient synthesis of a disulfide-containing protein through a batch cell-free system from 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 sear-ched here for translation conditions under which a model protein, a single-chain antibody variable fragment (scFv), could be synthesized into its active form. | Eur. J. Biochem. 270 4780-4786 2003 FEBS 2003 doi Efficient synthesis of a disulfide-containing protein through a batch cell-free system from wheat germ Takavasu Kawasaki1 MudeDDa D. Gouda1 Tatsuva Sawasaki1 2 Kazuvuki Takai1 2 and Yaeta Endo1 2 I 1 Cell-free Science and Technology Research Center CSTRC and 2Department of Applied Chemistry Faculty of Engineering Ehime University Matsuyama Japan 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