Yeast display

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The yeast display is a biochemical method with which recombinant proteins and peptides can be identified and evolved based on their binding properties ( affinity ) or based on their catalytic activity . For this purpose, members of a protein library are presented on the cell surface of yeast . The yeast display is an in vivo variant of the molecular display .

principle

The coupling of genotype and phenotype is essential in all molecular display systems . That is, the protein to be screened is coupled covalently or non-covalently by compartmentalization with the associated genetic information. The principle of the molecular display is based on the common occurrence of a protein and its coding DNA on or in a particle or a cell(in this case they are yeasts), whereby an interaction partner can be isolated from a mixture of transgenic yeasts, whose DNA is then also present, on the basis of the binding to an already present molecule. The DNA sequence corresponding to the recombinant surface protein is then extracted, amplified by PCR and sequenced by DNA sequencing . The amino acid sequence of the binding protein is then also known via the genetic code .

To identify a binding partner, gene libraries are cloned into the agap2 gene . For directed evolution, a fusion protein of the protein to be modified with the surface protein Aga2p is generated. Aga2p stands out from the glycocalyx of the yeast cell membrane and naturally serves for cell-cell contact when yeast mate. In the case of the fusion protein, the protein-binding part of the Aga2p was replaced by the protein to be changed or identified. The (affine) cells that bind to their target protein are selectively isolated by MACS or FACS .

The advantages of the yeast display over the in vitro and prokaryotic methods are the eukaryotic glycosylation of the presented protein and the protein quality control . Disadvantages are the glycosylations in yeast, which are similar to humans, but not identical, and the comparatively smaller gene library.

Applications

The yeast display is used for the selection and directed evolution of, among other things, recombinant antibodies .

Individual evidence

  1. ^ ET Boder, KD Wittrup: Yeast surface display for screening combinatorial polypeptide libraries. In: Nature Biotechnology . Volume 15, Number 6, June 1997, ISSN  1087-0156 , pp. 553-557, doi : 10.1038 / nbt0697-553 , PMID 9181578 .
  2. ↑ JM Weaver-Feldhaus, KD Miller, MJ Feldhaus, RW Siegel: Directed evolution for the development of conformation-specific affinity reagents using yeast display. In: Protein engineering, design & selection: PEDS. Volume 18, Number 11, November 2005, ISSN  1741-0126 , pp. 527-536, doi : 10.1093 / protein / gzi060 . PMID 16186140 ( PDF ).
  3. ↑ MJ Feldhaus, RW Siegel: Yeast display of antibody fragments: a discovery and characterization platform. In: Journal of immunological methods. Volume 290, Number 1-2, July 2004, ISSN  0022-1759 , pp. 69-80, doi : 10.1016 / j.jim.2004.04.009 , PMID 15261572 .
  4. ^ M. Feldhaus, R. Siegel: Flow cytometric screening of yeast surface display libraries. In: Methods in molecular biology (Clifton, NJ). Volume 263, 2004, ISSN  1064-3745 , pp. 311-332, doi : 10.1385 / 1-59259-773-4: 311 , PMID 14976374 .
  5. ↑ ET Boder, M. Raeeszadeh-Sarmazdeh, JV Price: engineering antibodies by yeast display. In: Archives of biochemistry and biophysics. Volume 526, Number 2, October 2012, ISSN  1096-0384 , pp. 99-106, doi : 10.1016 / j.abb.2012.03.009 , PMID 22450168 .
  6. ↑ MW Traxlmayr, C. Obinger: Directed evolution of proteins for Increased stability and expression using yeast display. In: Archives of biochemistry and biophysics. Volume 526, number 2, October 2012, ISSN  1096-0384 , pp. 174-180, doi : 10.1016 / j.abb.2012.04.022 , PMID 22575387 .