Method of producing secretable antibodies by expression in saccharomyces cerevisiae
The invention relates to a method for the production and non-covalent surface display of antibodies and derived fragments as well as molecule libraries based thereon on the surface of S. cerevisiae cells. The non-covalent manner of the surface display renders possible the selection of specific variants by means of high throughput screening and the subsequent switchable secretion of the selected binding molecule into the culture supernatant for biochemical characterization.
1. A method for producing a selected population of antibodies, by expression, secretion, and presentation of a mixed population of antibodies on the surface of yeast cells, the method comprising the following steps:
(a) providing host cells of the yeast species Saccharomyces cerevisiae that have been transfected with a first and a second nucleic acid molecule on expression plasmids, wherein the first nucleic acid molecule comprises a yeast GAL1 promoter and encodes a fusion protein, wherein the GAL1 promoter is capable of controlling the expression of said fusion protein as a function of cultivation conditions, and wherein said fusion protein comprises an alpha-agglutinin cell surface anchor protein and an Fc binding domain, and wherein the second nucleic acid molecule encodes an antibody of said mixed population of antibodies and is under the control of a permanently active promoter;
(b) cultivating said host cells of (a) in the presence of >10% w/v polyethylene glycol (PEG) having a molecular weight of >5,000 in the cultivation medium, under conditions whereby said mixed population of antibodies is simultaneously co-expressed in soluble form with said fusion protein, whereby said fusion protein is anchored to the surface of the host cell upon secretion, and whereby each host cell expresses an individual antibody of said mixed population of antibodies, and whereby said antibodies of said mixed population of antibodies are secreted from the host cells and bound in non-covalent form to said Fc binding domain of said fusion protein on the surface of each host cell,
(c) selecting and isolating a population of said host cells of (b) according to the affinity of individual antibodies in said mixed population of antibodies by binding a detection marker to said individual antibodies, wherein the detection marker is selective for said individual antibodies, and isolating the yeast cells which are bound to the detection marker,
(d) culturing said isolated host cells of (c) and expressing said individual antibodies selected in (c) under cultivation conditions wherein little or no expression of said fusion protein occurs and wherein the expressed antibodies from said second expression step are secreted from the yeast cell without binding to the surface of said isolated host cells of (c), and
(e) isolating the secreted antibodies expressed in (d) selected binding affinity.
2. The method according to claim 1 , wherein said fusion protein encoded by said first nucleic acid molecule comprises aga2p.
3. The method according to claim 2 , wherein said Fc binding domain is the protein A ZZ domain.
4. The method according to claim 3 , wherein said cell surface-anchored fusion protein is bound to a cell surface-displayed aga1p subunit.
5. The method according to claim 4 , wherein said permanently active promoter is the GAPDH promoter.
6. The method according to claim 1 , wherein said PEG comprises PEG8000 or a PEG with a molecular weight higher than 8000.
7. The method according to claim 1 , wherein said Saccharomyces cerevisiae is Saccharomyces cerevisiae strain EBY100.
8. The method according to claim 1 , wherein said first nucleic acid molecule and said second nucleic acid molecule are located on separate plasmids.
9. The method according to claim 8 , wherein at least one of said separate plasmids use pYDI as the starting plasmid.