Regulation of the display ratio of enzymes on the Saccharomyces cerevisiae cell surface by the immunoglobulin G and cellulosomal enzyme binding domains.
We constructed a novel cell surface display system to control the ratio of target proteins on the Saccharomyces cerevisiae cell surface, using two pairs of protein-protein interactions. One protein pair is the Z domain of protein A derived from Staphylococcus aureus and the Fc domain of human immunoglobulin G. The other is the cohesin (Coh) and dockerin (Dock) from the cellulosome of Clostridium cellulovorans. In this proposed displaying system, the scaffolding proteins (fusion proteins of Z and Coh) were displayed on the cell surface by fusing with the 3' half of alpha-agglutinin, and the target proteins fused with Fc or Dock were secreted. As a target protein, a recombinant Trichoderma reesei endoglucanase II (EGII) was secreted into the medium and immediately displayed on the yeast cell surface via the Z and Fc domains. Display of EGII on the cell surface was confirmed by hydrolysis of beta-glucan as a substrate, and EGII activity was detected in the cell pellet fraction. Finally, two enzymes, EGII and Aspergillus aculeatus beta-glucosidase 1, were codisplayed on the cell surface via Z-Fc and Dock-Coh interactions, respectively. As a result, the yeast displaying two enzymes hydrolyzed beta-glucan to glucose very well. These results strongly indicated that the proposed strategy, the simultaneous display of two enzymes on the yeast cell surface, was accomplished by quantitatively controlling the display system using affinity binding.
Department of Chemical Science and Engineering, Kobe University, Rokkodaicho, Nada-ku, Japan.
Junji
J
Ito
Akihiko
A
Kosugi
Tsutomu
T
Tanaka
Kouichi
K
Kuroda
Seiji
S
Shibasaki
Chiaki
C
Ogino
Mitsuyoshi
M
Ueda
Hideki
H
Fukuda
Roy H
RH
Doi
Akihiko
A
Kondo
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002480:Cellulase / Q000235:genetics / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D004798:Enzymes / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism; D047071:beta-Glucans / Q000378:metabolism; D000097722:Cohesins
D018797:Cell Cycle Proteins; D002480:Cellulase; D002868:Chromosomal Proteins, Non-Histone; D004798:Enzymes; D005947:Glucose; D007141:Immunoglobulin Fc Fragments; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D013205:Staphylococcal Protein A; D047071:beta-Glucans; D000097722:Cohesins