Yeast Display Systems

Abstract

The present invention relates to the field of protein display libraries and library screening. In preferred embodiments, the present invention provides a three component system for display comprising a cell surface molecule, an adapter molecule and a display molecule.

Description

FIELDThe present invention relates to the field of protein display libraries and library screening. More specifically, the present invention relates to the production of proteins for display on cell surfaces.BACKGROUNDProtein binding domains can be predicted from sequence data, however re-designing proteins with improved or altered binding affinities often requires testing of a number of variants of the re-designed protein. Currently the best method for obtaining proteins with desired binding affinities is to generate and screen a protein library including such variants that can include rationally redesigned proteins, randomly altered proteins, or a combination thereof. Libraries of many types of protein, such as immunoglobulins and scaffold proteins and receptors or receptor ligands have successfully been constructed and screened for binding affinity.There are many methods to screen libraries, but one of the most common methods is the phage display method, which comprises fusion of...

AI Technical Summary

Benefits of technology

The present invention provides methods and compositions for displaying modified polypeptides on host cells. The host cells have a cell surface molecule attached to the surface of the cell and a first nucleic acid encoding a display polypeptide comprising a modified polypeptide. An adapter molecule is contacted with the host cell and the display polypeptide, which binds specifically to the cell surface molecule and does not bind to the display molecule. The display polypeptide is then incubated under conditions wherein the host cell exports the display polypeptide outside the host cell. The invention allows for the display of a modified polypeptide on host cells with high efficiency and specificity. The host cells may be yeast cells or mammalian cells.

Problems solved by technology

However, one major drawback of this method is that target proteins that bind the library with very high affinity are not always identified because the conditions required to elute the bound phase usually denature the phage particle such that it becomes impossible to identify the protein of interest.

Another draw back of phage display libraries is the requirement that the target protein be immobilized on a solid surface, which can lead to difficulties in determining the actual affinity of a target protein for the phage display protein.

Furthermore, some proteins of interest require post-translational modifications, such as glycosylation, methylation, or disulfide binding, that cannot be achieved when expressed in a phage particle.

This method solves many of the drawbacks associated with phage display but has its own problems.

One problem with bacterial display is that the bacterial capsule can cause steric hindrance to proteins displayed on the bacterial surface.

Also, bacteria do not contain the machinery to properly fold eukaryotic proteins, so the protein of interest may not always be expressed within the bacterium.

Similar to the problem in phage, bacteria cannot provide post-translational modifications, like disulfide binding, to a eukaryotic protein.

One potential drawback of this system is that avidin non-specifically binds all biotin.

Another potential drawback is that avidin contains four binding sites, which may cause steric hindrance thus preventing the biotinylated protein library from binding to the cell surface bound avidin.

Additionally, this method contains the added complication of having to biotinylate the protein library within the yeast cell.

Such kits would be difficult to use in such a biotin / avidin display system.

The prior art lacks a simple, efficient system capable of specifically binding a secreted protein library using an adapter molecule that binds to the protein library and to the surface of a eukaryotic cell through different binding moieties.

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