An efficient and simple method to increase the level of displayed protein on the yeast cell surface.

Background: The development of oral vaccines using yeast surface display technology is an area of intensive study in vaccine development, but the protein level displayed on yeast surfaces is not currently high enough to obtain a robust immune response.

Methods: To address this issue, we established an efficient and simple method of increasing the level of displayed protein on the yeast cell surface. We used the single chain variable fragment (scFv) of an antibody against the infectious hematopoietic necrosis virus isolate Sn1203 as a target display protein. The yeast-derived scFv was first displayed on the yeast surface by galactose induction, and then Escherichia coli-derived scFv was also displayed on the same yeast via an artificial anchoring condition to increase the total scFv level on the yeast surface.

Results: The levels of yeast- and E. coli-derived scFv displayed on the yeast cell surface were analyzed by flow cytometry, western blotting, and fluorescent microscopy. The flow cytometry results indicated that when the cells were suspended in phosphate-buffered saline with 1mmol/L glutathione, 0.2mmol/L oxidized glutathione, and 5% dimethyl sulfoxide at 4°C for 6h, the E. coli-derived scFv protein was stably anchored to the yeast cell surface. The mean fluorescence intensity in these experiments, which is an indirect quantitative representation of the surface scFv expression, was three times higher in the treated cells than that in control cells. The western blotting results show two specific protein bands, the smaller of which was identified as the E. coli-derived scFv that was displayed on the yeast cell surface. Cell immunofluorescence is a more direct way to detect differentially produced proteins that are displayed on the yeast cell surface. The fluorescence microscopy results show that both fluorescence corresponding to the yeast-derived scFv and fluorescence corresponding to the E. coli-derived scFv can exist on the cell surface of same yeast cell. This confirms that the E. coli-derived scFv protein was successfully displayed on the yeast cell surface.

Conclusions: This method provides a rapid, simple, and high-efficiency strategy to increase the level of displayed protein on the yeast cell surface. Application of this technique may allow the yeast surface display system to be used to generate potential oral vaccines.