Construction of system for localization of target protein in yeast periplasm using invertase

We constructed a novel system for periplasmic localization of target proteins, using yeast external invertase (INV) as anchor protein, in which the C- or N-terminal of the target protein was fused to the invertase and the fusion proteins expressed under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (GAPDH). Unlike in conventional cell-surface display, the system enables the target fusion protein to localize in yeast periplasm in a free state. As a model, enhanced green fluorescence protein (EGFP) was localized in yeast periplasm using the new system. Yeast-periplasm localization of INV-EGFP and EGFP-INV fusion proteins was confirmed by fluorescence microscopy and immunoblotting: green fluorescence was observed at the cell outline and, in western blot analysis, most fusion proteins were detected in the cell-surface fraction, indicating that the fusion proteins had been transported to the cell-surface layer. In addition, in both C- and N-terminal fusion, invertase showed activity, indicating dimer formation. These results demonstrate that invertase is a useful anchor for localizing target protein in the yeast periplasm. (C) 2004 Elsevier B.V. All rights reserved.