Journal of Molecular Catalysis B: Enzymatic 28 (2004) 259–264 Construction of system for localization of target protein in yeast periplasm using invertase Takanori Tanino a , Takeshi Matsumoto b , Hideki Fukuda b , Akihiko Kondo a,∗ a Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japan b Division of Molecular Science, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japan Received 23 October 2003; received in revised form 27 November 2003; accepted 21 December 2003 Abstract 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. © 2004 Elsevier B.V. All rights reserved. Keywords: Saccharomyces cerevisiae; Periplasm; Invertase; Enhanced green fluorescence protein; Fusion protein 1. Introduction Cells that display enzymes on their surface can be used as whole-cell biocatalyst, and systems for display of het- erologous proteins on the yeast cell-surface have therefore recently been widely studied [1–5]. By combining the dis- played enzymes with a metabolic pathway, it is possible to catalyze sequential reactions. For example, a yeast display- ing endoglucanase II and -glucosidase on the cell-surface can produce ethanol directly from cellulosic materual [5]. Yeast-based cell-surface display systems have the advan- tages of safety, simplicity of genetic manipulation, and rigid- ity of cell-surface structure. In widely used cell-surface display systems, the tar- get protein is immobilized on the cell wall using the glycosylphosphatidylinositol (GPI)-anchor attachment sig- nal sequence [6] or the flocculation functional domain ∗ Corresponding author. Tel.: +81-78-803-6196; fax: +81-78-803-6206. E-mail address: kondo@cx.kobe-u.ac.jp (A. Kondo). of the Flo1 gene [3]. In addition to the cell wall, the periplasm, which is the space between the cell wall and the plasma membrane, is also advantageous for localizing target proteins. For instance, a whole-cell biocatalyst can achieve high activity through accumulation of enzyme in the periplasm layer, as such an arrangement circumvents the problem of substrate diffusion through the periplasm membrane. Moreover, by combining this novel periplasmic localization system with widely used cell-surface display systems, it will be possible to localize more enzymes in the cell-surface layer, namely outside of the plasma membrane. In the present study, we developed a novel cell-surface lo- calization system based on the invertase (-d-fructofurano- side fructohydrolase, E.C.3.2.1.26; INV) from Saccha- romyces cerevisiae. Since yeast external invertase localizes in the periplasm [7,8], it is possible to localize target pro- tein in the periplasm using invertase as an anchor protein. Enhanced green fluorescence protein (EGFP) was chosen as the target protein, as it allows localization to be visual- ized. EGFP was fused to the N- or C-terminal of invertase 1381-1177/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molcatb.2003.12.028