Evaluation of cell surface-displayed protein stability against simulated gastric fluid.
A molecular display technology that uses the displayed proteins on cell surfaces has many applications in microbiology and molecular biology. Here, we describe the resistance of displayed proteins to proteases using simulated gastric fluid (SGF), which included pepsin at pH 2. The displayed beta-glucosidase resisted pepsin digestion compared with secreted, free beta-glucosidase. In SDS-PAGE and Western blotting analysis, the secreted beta-glucosidase was immediately digested within 1 min following SGF treatment, although the displayed beta-glucosidase was stable for more than 60 min following SGF treatment. In addition, the residual activity of secreted beta-glucosidase was completely destroyed after 10 min SGF treatment. However, displayed beta-glucosidase retained 14% of its residual activity following the same treatment. These results clearly show that cell surface display technology using enzymes can reveal the protease resistance of a protein of interest under various conditions.
Horii
Ken
K
San-eigen F.F.I., Inc, 1-1-11 Sanwa-cho, Toyonaka, Osaka, 561-8588, Japan.
Adachi
Takashi
T
Tanino
Takanori
T
Tanaka
Tsutomu
T
Sahara
Hiroshi
H
Shibasaki
Seiji
S
Ogino
Chiaki
C
Hata
Yoji
Y
Ueda
Mitsuyoshi
M
Kondo
Akihiko
A
D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005750:Gastric Juice / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism*
D008565:Membrane Proteins; D011994:Recombinant Proteins; D001617:beta-Glucosidase