Evaluation of cell surface-displayed protein stability against simulated gastric fluid

Abstract

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.

Improvement in organophosphorus hydrolase activity of cell surface-engineered yeast strain using Flo1p anchor system

“…An OD 600 value of 1 = 0.313 mg dry cells/ml −1 (Horii et al. 2009). Next, 50 μl of 20 mM Paraoxon in methanol was added and reactions were carried out for 24 h at 30°C.…”

Development of Yeast Molecular Display Systems Focused on Therapeutic Proteins, Enzymes, and Foods: Functional Analysis of Proteins and its Application to Bioconversion

“…In an evaluation of pepsin digestibility assay in vitro using SGF against S. cerevisiae displayed protein on the cell surface [118], BGL1 from A. oryzae [100] was employed as a model protein, and digestibility of displayed BGL1 was evaluated using SGF. The digestibility of secreted BGL1 in SGF was first examined.…”

Methods of Protein Digestive Stability Assay - State of the Art

“…One of the simplest models of protein digestion is the use of the purified major enzymes of stomach (pepsin) and pancreas (trypsin and chymotrypsin) (Horii et al 2009;Huang et al 2010;Kamata et al 1982;Nielsen et al 1988;Stanic et al 2010;Yagami et al 2000), implemented either separately of subsequently. The products of the digestion are usually analyzed using SDS-PAGE with the separation range from 3 to 20-30 kDa (Stanic et al 2010;Yu et al 2011) or reverse-phase HPLC (Bublin et al 2008).…”

“…on the surface of a whole yeast cell. The aim of this study was to check the stability of expressed oral vaccine, and it has shown that indeed, a protein displayed on the yeast cell surface had an increased resistance to pepsin, possibly due to pepsin underwent steric hindrance and only digested the outermost side of the yeast cell wall protein (Horii et al 2009). …”