Phage Display Alternative: Invitrogen's Yeast Display Vectors

Aga2-fusion protein binding to yeast cell by Aga1. Image provided by Invitrogen. Phage display systems, reviewed in a recent issue of The Scientist,1 have proven enormously useful for the detection of protein-ligand interactions, as well as for the selection of variants with altered binding characteristics. However, limiting the utility of phage display systems are so-called expression biases against certain eukaryotic proteins, as well as the distinctly different protein processing mechanisms in prokaryotes as compared to eukaryotes. Invitrogen has introduced the pYD1 Yeast Display Vector Kit, which avoids both these problems and provides an alternative, easy screening method for peptide-ligand interactions. Based on the work of Eric Boder and Dane Wittrup (University of Illinois, Urbana), and offered for research purposes only under license from the University of Illinois, the Invitrogen yeast display system is centered around the vector pYD1, with which fusion proteins can be made to the membrane-associated alpha-agglutinin yeast adhesion receptor, a part of the mating type system. pYD1 carries the gene for the Aga2 subunit of the two-subunit receptor positioned to accept C-terminal fusions. Following transformation of a yeast host, expression of the aga2 gene, as well as its chromosomally based partner, the aga1 gene, can be induced with galactose (both genes are under the control of a gal1 promoter)--a nice feature when working with potentially toxic proteins. The Aga1 protein is covalently linked into the cell wall by phosphatidyl inositol glycan linkages, and is the tether that binds the Aga2 fusion protein to the yeast cell, once it self-assembles onto Aga1 by disulfide bond linkages. In the original description of the system,2 Boder and Wittrup created a fusion protein with an antifluorescein single-chain Fv antibody fragment and a c-myc epitope attached to the C-terminus of the Aga2 protein. Double label experiments with anti-c-myc monoclonal antibody and FITC-dextran demonstrated the accessibility of the epitopes carried on the fusion protein to large macromolecules. In addition, the researchers went on to select from a modest-size library of 5 x 105 mutated scFv genes, several variants of which displayed significantly different binding constants for FITC-dextran (two-to threefold longer on time), demonstrating the utility of the yeast system as a screen. Since the original description of the system, the antifluorescein antibody chain has been affinity matured to femtomolar affinity by yeast display (Wittrup, personal communication), resulting in a significantly longer half-life (12 days as opposed to 5 minutes). In addition, the T-cell receptor, largely refractory to analysis due to the difficulty in solubilizing the protein, has been the object of study in yeast display: Yeast display has been used to affinity mature an anti-T-cell receptor antibody, to activate T cells via multivalent display of anti-T-cell receptor scFvs, to create the first single-chain T-cell receptor display library, and to stabilize and improve expression of single-chain T-cell receptors.3 In addition to having inducible expression, the pYD1 vector also has several C-terminal tags (V5 and 6xHis) to facilitate the detection of fusion proteins and an N-terminal tag and linker to optimize display. It also sports a large multiple cloning site as well as TRP1 auxotropic marker and CEN/ARS origin for growth and selection in yeast host cells. The Invitrogen kit comes with the vector, forward and reverse primers for verifying the fusion, and the host yeast (S. cerevisiae) strain EBY100. Antibodies against the tags V5 and His are available separately. D. Swanson, "The ties that bind," The Scientist, 13[6]:22, March 15, 1999. E.T. Boder and K.D. Wittrup, "Yeast surface display for screening combinatorial polypeptide libraries," Nature Biotechnology, 15:553-7, 1997. Eric V. Shusta et al., "Biosynthetic polypeptide libraries," Current Opinion in Biotechnology, 10:117-122, 1999. The author, Laura De Francesco, can be contacted at neumann@earthlink.net.

Phage Display Alternative: Invitrogen's Yeast Display Vectors

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