A yeast display system for engineering functional peptide-MHC complexes
| Autor: | David M. Kranz, Phillip D. Holler, Susan E. Brophy |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
T-Lymphocytes
Immunology Antigen presentation Genetic Vectors Receptors Antigen T-Cell Saccharomyces cerevisiae Yeast display Biology In Vitro Techniques Major histocompatibility complex Protein Engineering Antibodies Mice Antigen Peptide Library MHC class I Immunology and Allergy Cytotoxic T cell Animals Peptide library Histocompatibility Antigens Class I H-2 Antigens MHC restriction Molecular biology Biochemistry biology.protein Immunologic Techniques Directed Molecular Evolution Oligopeptides |
| Zdroj: | Journal of immunological methods. 272(1-2) |
| ISSN: | 0022-1759 |
| Popis: | In a cellular immune response, antigenic peptides derived by intracellular processing of foreign pathogens are bound to the class I major histocompatability complex (MHC I) and presented to CD8(+) cytotoxic T cells. Although the crystal structures of several different MHC products have been solved, many MHC molecules, including some associated with diseases, have not been amenable to biochemical and structural studies. The variability in this success is based largely on the fact that peptide-MHC complexes vary extensively in their stability. These properties also are intimately tied to the biological activity of the complexes. The ability to apply the techniques of directed evolution to this system in order to engineer stable complexes has been complicated by the trimeric structure of peptide-MHC complexes, requiring association of three polypeptides: the heavy chain, beta2-microglubulin (beta2m), and a short peptide. We show here that single-chain forms of peptide-MHC complexes can be expressed as Aga-2 fusions on the surface of yeast. Three different complexes, SIYRYYGL-K(b)-beta2m (SIYR-K(b)), EQYKFYSV-K(b)-beta2m (dEV8-K(b)), and SIINFEKL-K(b)-beta2m (OVA-K(b)), were expressed on yeast and detected by flow cytometry with a conformation-specific anti-K(b) antibody (B.8.24.3). In addition, yeast displaying K(b) loaded with exogenous SIYR and OVA peptides were recognized by a high-affinity T cell receptor that is specific for SIYR-K(b) and by an antibody (25.D1-16) that is specific for OVA-K(b), respectively. Finally, yeast that display the SIYRYYGL-K(b) also directly stimulated CD69 up-regulation on naive 2C T cells. Hence, yeast display represents a technology that can be used for directed evolution of any of the components of the trimeric pep-MHC complex. |
| Databáze: | OpenAIRE |
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