High-efficiency recovery of target cells using improved yeast display system for detection of protein-protein interactions.
We constructed a high-throughput screening (HTS) system for target cells based on the detection of protein-protein interactions by flow cytometric sorting due to the improvement in the yeast cell surface display system. Interaction model proteins, which are the ZZ domain derived from Staphylococcus aureus and the Fc part of human immunoglobulin G (IgG), were displayed on the yeast cell surface. We achieved a rapid and enhanced expression of these proteins as a result of adopting an appropriate yeast strain and a suitable promoter. The displayed ZZ domain had an ability to bind to rabbit IgG and the displayed Fc part to protein A. These were confirmed by flow cytometry and fluorescence microscopy. Furthermore, the cells displaying the ZZ domain or Fc part were isolated from the model libraries constructed by mixing the control yeast cells with the target yeast cells. The ratio of the target cells was increased from 0.0001% to more than 70% by two cycles of cell sorting. These results indicate that we can achieve a rapid and highly efficient isolation method for the target cells with FACSCalibur and that this method will further extend the application of flow cytometric sorting to library selections.
Fukuda
Nobuo
N
Department of Chemical Science and Engineering, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, 657-8501, Japan.
Ishii
Jun
J
Shibasaki
Seiji
S
Ueda
Mitsuyoshi
M
Fukuda
Hideki
H
Kondo
Akihiko
A
D000818:Animals; D002469:Cell Separation; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D015723:Gene Library; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013211:Staphylococcus aureus / Q000737:chemistry
D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A