Design of solubly expressed miniaturized SMART MHCs

The precise recognition of specific peptide-major histocompatibility complex (pMHC) complexes by T cell receptors (TCRs) plays a key role in infectious disease, cancer, and autoimmunity. A critical step in many immunobiological studies is the identification of T cells expressing TCRs specific to a given pMHC antigen. However, the intrinsic instability of empty class-I MHCs limits their soluble expression in <i><i>Escherichia coli</i></i> and makes it very difficult to characterize even a small fraction of possible pMHC/TCR interactions. To overcome this limitation, we designed small proteins which buttress the peptide binding groove of class I MHCs, replacing β2-microglobulin (β2m) and the heavy chain α3 domain, and enable soluble and partially soluble expression in <i><i>E. coli</i></i> of H-2D<sup>b</sup> and A*02:01, respectively. We demonstrate that these soluble, monomeric, antigen-receptive, truncated (SMART) MHCs retain both peptide- and TCR-binding specificity and that peptide-bound structures of both allomorphs are similar to their full-length, native counterparts. With extension to the majority of HLA alleles, SMART MHCs should be broadly useful for probing the T cell repertoire in approaches ranging from yeast display to T cell staining.

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Publication Details

Journal
Proceedings of the National Academy of Sciences
Published
2026-01-02
DOI
https://doi.org/10.1073/pnas.2505932123
Primary Topic
T-cell and B-cell Immunology
Type
article
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article

Design of solubly expressed miniaturized SMART MHCs

Peter Nygren, Kevin M. Jude, D. Baker, Nikolaos G. Sgourakis et al.
Proceedings of the National Academy of Sciences
T-cell and B-cell Immunology
article

Design of solubly expressed miniaturized SMART MHCs

Peter Nygren, Kevin M. Jude, D. Baker, Nikolaos G. Sgourakis, Aza Allen, Chunyu Wang, Michael C. Jewett, Paul M. Levine, Inna Goreshnik, Laura Guerrero, Apala Chaudhuri, Xinting Li, Hao Yuan Kueh, Xiaojing Chen, Amarachi E. Onwuka, Chun Kim, W Thomas White, Julia E. Bonzanini, Adnane Achour, Renhua Sun, Xiao Han, Hua Bai, Nan Wang, Yi Sun, K. Christopher Garcia
article en

Abstract

The precise recognition of specific peptide-major histocompatibility complex (pMHC) complexes by T cell receptors (TCRs) plays a key role in infectious disease, cancer, and autoimmunity. A critical step in many immunobiological studies is the identification of T cells expressing TCRs specific to a given pMHC antigen. However, the intrinsic instability of empty class-I MHCs limits their soluble expression in <i><i>Escherichia coli</i></i> and makes it very difficult to characterize even a small fraction of possible pMHC/TCR interactions. To overcome this limitation, we designed small proteins which buttress the peptide binding groove of class I MHCs, replacing β2-microglobulin (β2m) and the heavy chain α3 domain, and enable soluble and partially soluble expression in <i><i>E. coli</i></i> of H-2D<sup>b</sup> and A*02:01, respectively. We demonstrate that these soluble, monomeric, antigen-receptive, truncated (SMART) MHCs retain both peptide- and TCR-binding specificity and that peptide-bound structures of both allomorphs are similar to their full-length, native counterparts. With extension to the majority of HLA alleles, SMART MHCs should be broadly useful for probing the T cell repertoire in approaches ranging from yeast display to T cell staining.

Proceedings of the National Academy of SciencesVol. 123(1)
Karolinska University Hospital (SE), Children's Hospital of Philadelphia (US), University of Washington (US), Science for Life Laboratory (SE), Bioengineering Center (RU), Center for Genomic Science (IT), Protein Research Foundation (JP), PDL BioPharma (United States) (US), University of Pennsylvania (US), Stanford University (US)
Openalex Percentile: Top 99%
T-cell and B-cell Immunology
DOIOpen Access PDFOpenAlex
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