Long anchor using Flo1 protein enhances reactivity of cell surface-displayed glucoamylase to polymer substrates.
We investigated the influence of anchor length on the reactivity to polymer substrate of enzyme displayed on yeast cell surfaces. Using various lengths [42, 102, 146, 318, 428, and 1,326 amino acids (aa)] of the C-terminal region of the Saccharomyces cerevisiae Flo1 protein (Flo1p), which plays a major role in yeast flocculation, six display systems with various anchor lengths were constructed. In these systems, the target protein was displayed on the yeast cell surface under the control of the 5'-upstream region of the isocitrate lyase gene of Candida tropicalis ( UPR-ICL). Cell-surface display of Rhizopus oryzae glucoamylase by these systems was induced and confirmed in all systems by immunofluorescence microscopy and immunoblotting. Flow-cytometer measurement of the fluorescence intensity of immunofluorescence-labeled yeast cells displaying glucoamylase indicated that glucoamylase displayed with longer anchors, especially those of 428 and 1,326 aa in length, had higher reactivity to antibodies. The reactivity of starch to displayed glucoamylase, which was evaluated by plate assay, increased with anchor length, as did the cell growth-rate in starch-containing medium. These results indicate that cell-surface display systems using 428- and 1,326-aa length anchors of Flo1p are effective for the display of enzymes on the outer surface of yeast cells.
Sato
N
N
Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Japan.
Matsumoto
T
T
Ueda
M
M
Tanaka
A
A
Fukuda
H
H
Kondo
A
A
D003001:Cloning, Molecular; D005427:Flocculation; D005087:Glucan 1,4-alpha-Glucosidase / Q000378:metabolism*; D037241:Mannose-Binding Lectins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D011108:Polymers / Q000378:metabolism; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013379:Substrate Specificity
C087578:FLO1 protein, S cerevisiae; D037241:Mannose-Binding Lectins; D008565:Membrane Proteins; D011108:Polymers; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D005087:Glucan 1,4-alpha-Glucosidase