Quantitative evaluation of the enhanced green fluorescent protein displayed on the cell surface of Saccharomyces cerevisiae by fluorometric and confocal laser scanning microscopic analyses.
The number of foreign protein molecules expressed on the cell surface of the budding yeast Saccharomyces cerevisiae by cell surface engineering was quantitatively evaluated using enhanced green fluorescent protein (EGFP). The emission from EGFP on the cell surface was affected by changes in pH. The amount of EGFP on the cell surface, displayed as alpha-agglutinin-fusion protein under control of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, was determined at the optimum pH of 7.0. The fluorometric analysis and the image analysis by confocal laser scanning microscopy (CLSM) showed a similar number of molecules displayed on the cell surface, demonstrating that 10(4)-10(5) molecules of alpha-agglutinin-fused molecules per cell were expressed. Furthermore, the amount of fluorescent protein expressed on cells harboring a multicopy plasmid was three to four times higher than that on cells harboring the gene integrated into the genome.
Shibasaki
S
S
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan.
Ueda
M
M
Iizuka
T
T
Hirayama
M
M
Ikeda
Y
Y
Kamasawa
N
N
Osumi
M
M
Tanaka
A
A
D017360:Arabidopsis / Q000254:growth & development*; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D005069:Evaluation Studies as Topic; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D017931:DNA Primers; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases