A yeast display system for engineering functional peptide-MHC complexes

Brophy, Susan; Holler, Phillip D.; Kranz, David M.

doi:10.1016/s0022-1759(02)00439-8

Cited by 30 publications

(24 citation statements)

References 41 publications

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“…Based on our studies with single-chain TCRs (36,37,40,44,51), we have predicted that such displayed proteins are likely to exhibit higher levels of expression and folding as soluble constructs. Our previous study also showed that a single-chain K b /␤2m construct could be displayed on yeast (13), but efforts to engineer the construct by directed evolution were not attempted. In this report, we used a process of random mutagenesis and selection using yeast display to identify mutants of the class I molecule L d that were expressed at high levels on the surface of yeast.…”

Section: Discussionmentioning

confidence: 99%

“…Although many proteins have been amenable to engineering by in vitro directed evolution and peptide-MHC complexes have been displayed on phage (10 -12) or yeast (13), few studies have used directed evolution approaches to engineer more stable MHC molecules (14 -16). In this regard, H-2L d has been shown through both biochemical and structural studies to be less stable than many other MHC complexes.…”

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confidence: 99%

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Engineering and Characterization of a Stabilized α1/α2 Module of the Class I Major Histocompatibility Complex Product Ld

Jones

Brophy

Bankovich

et al. 2006

Journal of Biological Chemistry

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Engineering and Characterization of a Stabilized α1/α2 Module of the Class I Major Histocompatibility Complex Product Ld

Jones

Brophy

Bankovich

et al. 2006

Journal of Biological Chemistry

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“…As a eukaryotic organism, yeast is better than E. coli at expressing complex eukaryotic proteins, such as MHC molecules, antibodies, and ␣␤TCRs (26)(27)(28)(29). Frequently, however, considerable engineering and mutation of these molecules have been required for efficient surface display, and the use of yeast surface MHC display for identifying TCR peptide mimotopes has not been reported.…”

Section: Discussionmentioning

confidence: 99%

Using a baculovirus display library to identify MHC class I mimotopes

Wang

Rubtsov

Heiser

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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We have developed a baculovirus-based display system for identifying antigen mimotopes for MHC class I-specific T cells. The mouse MHC class I molecule, D d , was displayed on baculovirusinfected insect cells with a library of 9-and 10-mer peptides tethered via a flexible linker to the N terminus of ␤2 microglobulin. As a test case, the library was screened by flow cytometry by using a multimeric fluorescent ␣␤TCR from a mouse T cell specific for D d plus an unknown self peptide. A mimotope was identified that, when bound to D d , stimulated the T cell to secret IL-2. The sequence of the mimotope was used to identify a self peptide present in a mouse protein, Spin. The Spin peptide, when complexed with D d , also activated the T cell. This technique should be generally useful in identifying and manipulating MHC class I peptide mimotopes and epitopes.T cell receptor ͉ peptide ͉ epitope T he identification of peptide antigen epitopes is an important step in isolating and modulating class I MHC (MHCI)-restricted T cells involved in protective and pathological immune responses. The identification of MHCI peptide antigens is difficult in autoimmunity and cancer, because, in these cases, the peptide antigen may be derived from any of the host's proteins expressed in the target tissue. As a consequence, although there are many candidate antigens, in almost no case is the T cell epitope(s) involved in human autoimmunity definitively known. Likewise, despite years of work, the list of tumor-associated antigens with potential for use in immunotherapy is still quite small and concentrated in a few types of cancer.Many approaches have been taken to identify MHCI peptide epitopes derived from unknown self proteins. One approach has been to identify peptide mimotopes in peptide libraries. Mimotopes differ in sequence from the unknown peptide epitope, but they nevertheless bind to the appropriate MHCI molecule and are recognized by the specific CD8 ϩ T cell (1-3). They can be used to track the T cell in vivo and for immune modulation of the T cell response. Sometimes, the sequence of the mimotope can be used to deduce the sequence of the epitope (4).Peptide display libraries have been very powerful tools in other situations (reviewed in refs. 5-7), but they are not widely used to identify T cell mimotopes, because the peptide can be identified and used to enrich the library only when it is properly associated with the appropriate MHC molecule. We recently developed baculovirus-infected insect cells as a display platform for class II MHC (MHCII) molecules covalently bound to a library of potential peptide mimotopes (8). ''Fishing'' in this library with soluble fluorescent T cell receptors, we were able to identify peptide mimotope͞MHC complexes that bound to the soluble receptors and stimulated T cells bearing the same receptors. In this present study, we have adapted this method for use with MHCI. In this case, we expressed the peptide library covalently bound to ␤2 microglobulin (␤2m) paired with membrane anchored MHCI D d he...

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“…Considering that in nature the combinatorial antibody diversity is a result of somatic recombination, it is not surprising that directed evolution can be a powerful and practical tool for the creation of high-affinity antibodies in vitro. Techniques such as surface display facilitate high-throughput selection for desired activity (32,62,85,124,143,295,308). Recombination of phagedisplayed, low-affinity immunoglobulin M antibodies resulted in variants with increased affinity of several orders of magnitude in just two rounds of evolution (85).…”

Section: Directed Evolution Of Pharmaceuticalsmentioning

confidence: 99%

Laboratory-Directed Protein Evolution

Yuan

Kurek

English

et al. 2005

Microbiol Mol Biol Rev

175

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Systematic approaches to directed evolution of proteins have been documented since the 1970s. The ability to recruit new protein functions arises from the considerable substrate ambiguity of many proteins. The substrate ambiguity of a protein can be interpreted as the evolutionary potential that allows a protein to acquire new specificities through mutation or to regain function via mutations that differ from the original protein sequence. All organisms have evolutionarily exploited this substrate ambiguity. When exploited in a laboratory under controlled mutagenesis and selection, it enables a protein to “evolve” in desired directions. One of the most effective strategies in directed protein evolution is to gradually accumulate mutations, either sequentially or by recombination, while applying selective pressure. This is typically achieved by the generation of libraries of mutants followed by efficient screening of these libraries for targeted functions and subsequent repetition of the process using improved mutants from the previous screening. Here we review some of the successful strategies in creating protein diversity and the more recent progress in directed protein evolution in a wide range of scientific disciplines and its impacts in chemical, pharmaceutical, and agricultural sciences

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A yeast display system for engineering functional peptide-MHC complexes

Cited by 30 publications

References 41 publications

Engineering and Characterization of a Stabilized α1/α2 Module of the Class I Major Histocompatibility Complex Product Ld

Engineering and Characterization of a Stabilized α1/α2 Module of the Class I Major Histocompatibility Complex Product Ld

Using a baculovirus display library to identify MHC class I mimotopes

Laboratory-Directed Protein Evolution

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