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Yeasts are single-celled organisms, but unlike bacteria, they are eukaryotes (cells with a nucleus). Cell signaling in yeast is similar to signaling in other eukaryotic cells. A ligand, such as a protein or a small molecule released from a yeast cell, attaches to a receptor on the cell surface. The binding stimulates second-messenger kinases to activate or inactivate transcription factors that further regulate gene expression. Many of the yeast intracellular signaling cascades have similar...
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Enzymatic Modification and Flow Cytometry Assessment of Yeast Surface Displayed Proteins
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A yeast display system for engineering functional peptide-MHC complexes.

Susan E Brophy1, Phillip D Holler, David M Kranz

  • 1Department of Biochemistry, University of Illinois, 600 S. Matthews Avenue, Urbana 61801, USA.

Journal of Immunological Methods
|December 31, 2002
PubMed
Summary
This summary is machine-generated.

Yeast display technology enables the creation of stable single-chain peptide-MHC class I complexes. This method facilitates directed evolution for engineering these crucial immune system components.

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Area of Science:

  • Immunology and Structural Biology

Background:

  • Cellular immune responses involve presenting foreign peptides via Major Histocompatibility Complex class I (MHC I) molecules to CD8(+) T cells.
  • Structural studies of many MHC I molecules are challenging due to their complex trimeric structure and variable stability, hindering directed evolution efforts.
  • Understanding and engineering peptide-MHC (pMHC) complexes is vital for immunology and disease research.

Purpose of the Study:

  • To develop a novel method for expressing stable, single-chain peptide-MHC class I complexes.
  • To demonstrate the utility of yeast display for directed evolution of pMHC components.
  • To enable engineering of pMHC complexes for improved stability and biological function.

Main Methods:

  • Engineered single-chain peptide-MHC class I complexes as Aga-2 fusions for display on yeast.
  • Expressed three distinct pMHC complexes: SIYRYYGL-K(b)-beta2m (SIYR-K(b)), EQYKFYSV-K(b)-beta2m (dEV8-K(b)), and SIINFEKL-K(b)-beta2m (OVA-K(b)).
  • Utilized flow cytometry with specific antibodies and T cell receptor recognition assays to validate yeast display and complex integrity.

Main Results:

  • Successfully expressed functional pMHC I complexes on the yeast surface, confirmed by flow cytometry and specific antibody binding.
  • Yeast-displayed complexes were recognized by cognate T cell receptors and specific antibodies, validating their correct folding and peptide loading.
  • Yeast displaying SIYRYYGL-K(b) directly stimulated CD69 upregulation on naive 2C T cells, demonstrating biological activity.

Conclusions:

  • Yeast display is a viable technology for producing single-chain peptide-MHC class I complexes.
  • This platform supports the directed evolution of all components within the trimeric pMHC complex.
  • The developed method advances the engineering of stable and functional pMHC molecules for immunological applications.