Result

Fig. 1 Immunofluorescence labeling of transformed cells: green fluorescence corresponds to cells labeled with Mouse Anti HA-Tag Polyclonal antibody and (Fluorescein (FITC)–conjugated Affinipure Goat Anti-Mouse IgG(H+L), and red fluorescence corresponds to cells labeled with Rabbit Anti Flag-Tag Polyclonal antibody and Goat Anti Rabbit RPE Polyclonal antibody.

Immunofluorescence analysis of β-glucosidase surface display Pichia pastoris using flow cytometry.

We fused HA-tag with N-terminal of bglX gene on ZXFα-bglX vector. Label cells with Mouse Anti HA-Tag Polyclonal antibody and Fluorescein (FITC)–conjugated Affinipure Goat Anti-Mouse IgG(H+L). Result of flow cytometry analysis is as follows: fluorescence intensity x-mean value of Pichia pastoris with pPICZα empty vector is 0.306, with bglX-ZXFα is 23.4.

Fig. 2-1 Left: pPICZα negative control (x-mean: 0.306/1.17); Right: bglX (x-mean: 23.4/23.6)

Immunofluorescence analysis of endoxylanase surface display Pichia pastoris using flow cytometry.

We fused HA-tag with N-terminal of xyn gene on ZXFα-xyn vector. Label cells with Mouse Anti HA-Tag Polyclonal antibody and Fluorescein (FITC)–conjugated Affinipure Goat Anti-Mouse IgG(H+L). Result of flow cytometry analysis is as follows: fluorescence intensity x-mean value of Pichia pastoris with pPICZα empty vector is 0.62, with xyn-ZXFα is 67.

Fig. 2-2 Left: pPICZα negative control (x-mean: 0.62/4.31); Right: xyn (x-mean: 67/67.6)

Immunofluorescence analysis of β-xylosidase Ruxyn surface display Pichia pastoris using flow cytometry.

We fused FLAG-tag with N-terminal of Ruxyn gene on Ruxyn-pPIC9k vector. Label cells with Rabbit Anti Flag-Tag Polyclonal antibody and Goat Anti Rabbit RPE Polyclonal antibody. Result of flow cytometry analysis is as follows: fluorescence intensity x-mean value of Pichia pastoris with pPIC9k empty vector is 1.14, with Ruxyn-pPIC9k is 8.56.

Fig. 2-3 Left: pPIC9k negative control (x-mean: 1.14/13.4); Right: Ruxyn (x-mean: 8.56/19.9)

Immunofluorescence analysis of endoglucanase cel5A surface display Pichia pastoris using flow cytometry.

We fused FLAG-tag with N-terminal of cel5A gene on cel5A-pPIC9k vector. Label cells with Rabbit Anti Flag-Tag Polyclonal antibody and Goat Anti Rabbit RPE Polyclonal antibody. Result of flow cytometry analysis is as follows: fluorescence intensity x-mean value of Pichia pastoris with pPIC9k empty vector is 1.14, with cel5A-pPIC9k is 5.55.

Fig. 2-4 Left: pPIC9k negative control (x-mean: 1.14/13.4); Right: cel5A (x-mean: 5.55/16.5)