Immobilization of Saccharomyces cerevisiae cells to protein G-Sepharose by cell wall engineering.
In this work, we explored the possibility of using the targeting of a heterologous protein to the cell wall of Saccharomyces cerevisiae, by fusing it to a cell wall protein, to construct yeast strains whose cells display on their surface proteins that bind to a matrix, so as to achieve the immobilization of the whole cells. With this aim, we created a gene fusion that comprises the region responsible for attachment of a cell wall protein to the cell wall, and the IgG binding region of staphylococcal protein A, and expressed it in the mnn1mnn9 strain of S. cerevisiae. The surface display of the protein A-Icwp fusion protein was positively monitored; however, direct immobilization of the cells on an IgG-Sepharose matrix did not produce the expected results, probably due to the fusion protein not being completely exposed on the surface of the cells. To solve this problem we incubated the cells first with rabbit preimmune serum and then with goat anti-rabbit IgGs, so as to create a complex (yeast cell-protein A-rabbit IgG-goat IgG). Cells treated in this way were successfully immobilized on a protein G-Sepharose matrix, due to the binding properties of goat IgGs to streptococcal protein G.
Andrés
Isabel
I
Unidad de Microbiología, Fac. Farmacia, Universitat de Valencia, Burjassot, Spain.
Zueco
Jesús
J
Parascandola
Palma
P
D000818:Animals; D002473:Cell Wall / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018914:Cells, Immobilized; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005818:Genetic Engineering / Q000379:methods*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D012685:Sepharose / Q000031:analogs & derivatives*; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism
C105281:CCW14 protein, S cerevisiae; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D013205:Staphylococcal Protein A; C036210:immunoglobulin G-sepharose; D012685:Sepharose