5′ Flo1P Signal sequence FL FS N N a Secretion signal sequence of α-factor of S. cerevisiae 5′ GPI anchor attachment signal sequence GAPDH Promoter Target protein Target gene 3′-Half of α-agglutinin Translation 3′-Half of α-agglutinin α-Agglutinin fusion protein b c Secretion signal sequence of Flo1p 5′ UPR-ICL Promoter Translation Flocculation domain Flo1p fusion protein 1 834 3261 Proposed flocculation 3′ C GPI anchor 3′ Target gene C Target protein 4563 3′ Target gene C 656 parathion (Tuovinen et al. 1994). We previously constructed a yeast strain that displayed OPH from Flavobacterium species; the cell surface-engineered yeast-displayed OPH using glycosylphosphatidylino- sitol (GPI) anchor system (Takayama et al. 2006). Details of this system are shown in Fig. 1a. Yeasts displaying OPH exhibited greater activity than Esch- erichia coli displaying OPH using the ice nucleation protein (INP) anchor system (Shimazu et al. 2001). However, it is possible that OPH activity might be suppressed in yeast strains when using GPI anchor system. Because the OPH active site is located near the C-terminus, the 30-half of a-agglutinin of the GPI anchor might be sterically hindered (Matsumoto et al. 2002). Therefore, we adopted an optimal anchor system for the location of the OPH active site. In this study, yeasts displaying OPH using Flo1p anchor system were constructed. Flo1p is a lectin-like cell-wall protein in Saccharomyces cerevisiae. Detail of Flo1p are shown in Fig. 1b. Using Flo1p anchor system, a recombinant lipase having its active site near the C-terminus was displayed on the yeast and it showed higher activity than that obtained using GPI anchor system. Using Flo1p anchor system, we attempted to construct OPH-displaying yeasts that exhibited greater activity compared with that obtained using GPI anchor system. Materials and methods Strains and media E. coli strain, DH5a [F-, endA1, hsdR17 (rK-, mK?), supE44, thi-l, k-, recA1, gyrA96, DlacU169 (u80lacZ DM15)], was used as a host cell for recombinant DNA manipulation. Saccharomyces cerevisiae MT8-1 (MATa, ade, his3, leu2, trp1, ura3) (Tajima et al. 1985), was used to display OPH from Flavobacterium species on its cell surface. LB medium with 50 lg ampicillin ml-1 was used for bacterial growth and plasmid ampliﬁcation. The yeast cells were grown in YPD medium [1% (w/v) yeast extract, 2% (w/v) polypeptone, and 2% (w/v) glucose]. Yeast recombinant transformants were selected on plates containing SD-W medium [0.67% (w/v) yeast nitrogen base without amino acids (Difco, MI, USA), 2% (w/v) glucose, 0.002% (w/v) adenine sulfate, 0.002% (w/v) L-histidine- HCl, 0.003% (w/v) L-leucine, and 0.002% (w/v) uracil]. 123 Biotechnol Lett (2010) 32:655–659 Proposed flocculation functional domain 4341 3297 N Translation Flocculation domain Target protein Flo1p (FL/FS) fusion protein Fig. 1 The a-agglutinin fusion gene and Flo1p fusion gene for display of protein on the yeast cell surface. a a-Agglutinin fusion protein. The secretion signal sequence of a-agglutinin and the GPI anchor attachment signal sequence are genetically fused to the C-terminal region of the target protein. b Flo1p fusion protein. The N-terminal regions of target proteins such as OPH are fused to the Flo1p ﬂocculation functional domain. c Design of Flo1p (FL/FS) fusion gene for display of protein on the yeast cell surface HEPES was added to the liquid SD-W medium at a concentration of 200 mM (pH 7.0). Cell growth in the culture broth was measured based on absorbance at 600 nm. Construction of plasmids for cell surface display Plasmids for OPH display using Flo1p anchor system were constructed as follows. The gene encoding OPH was ampliﬁed using PCR with pWM513 (Takayama et al. 2006) as the template (Fig. 2). Two forward