Construction of aPichia pastorisCell-Surface Display System Using Flo1p Anchor System

Tipo de material: Texto

TextoSeries ; Biotechnology Progress, 22(4), p.989-993, 2006Trabajos contenidos:

Tanino, T
Fukuda, H
Kondo, A

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Resumen: APichia pastoriscell-surface display system was constructed using a Flo1p anchor system, which was developed inSaccharomyces cereVisiae. The lipase from Rhizopus oryzaewith 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 immunolabeledP. pastoriscells 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 ofP. pastoriscells was affected by the methanol concentration for the induction phase. Surprisingly, the activity of lipase displayed on the cells incubated at 60 °C was not only stable but also increased to about 6.5 times the initial value after 4 h incubation.

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APichia pastoriscell-surface display system was constructed using a Flo1p anchor system, which was developed inSaccharomyces cereVisiae. The lipase from Rhizopus oryzaewith 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 immunolabeledP. pastoriscells 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 ofP. pastoriscells was affected by the methanol concentration for the induction phase. Surprisingly, the activity of lipase displayed on the cells incubated at 60 °C was not only stable but also increased to about 6.5 times the initial value after 4 h incubation.

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