Construction of a Pichia pastoris cell-surface display system using Flo1p anchor system.
A Pichia pastoris cell-surface display system was constructed using a Flo1p anchor system, which was developed in Saccharomyces cerevisiae. The lipase from Rhizopus oryzae with a pro sequence (ProROL) was used as the model protein and was genetically fused to the anchor consisting of amino acids 1-1099 of Flo1p (FS anchor). The resulting fusion protein FSProROL was expressed under the control of the alcohol oxidase 1 promoter (pAOX1). The fluorescence microscopy of immunolabeled P. pastoris cells revealed that ProROL was displayed on the cell surface, and Western blot analysis revealed that the fusion protein FSProROL was noncovalently attached to the cell wall and highly glycosylated. The lipase activity of P. pastoris cells was affected by the methanol concentration for the induction phase. Surprisingly, the activity of lipase displayed on the cells incubated at 60 degrees C was not only stable but also increased to about 6.5 times the initial value after 4 h incubation.
Tanino
Takanori
T
Division of Molecular Science and Material Engineering, Graduate School of Science and Technology, and Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, Nada-ku, Japan.
Fukuda
Hideki
H
Kondo
Akihiko
A
D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation; D008049:Lipase / Q000737:chemistry* / Q000378:metabolism; D037241:Mannose-Binding Lectins; D008856:Microscopy, Fluorescence / Q000379:methods; D010843:Pichia / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D012233:Rhizopus / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors
C087578:FLO1 protein, S cerevisiae; D037241:Mannose-Binding Lectins; D029701:Saccharomyces cerevisiae Proteins; D008049:Lipase