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In Vitro Reconstitution of the MHC Class I Peptide-Loading Complex

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Antigen Processing

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 960))

Abstract

The stability of the MHC (major histocompatibility complex) class I peptide repertoire is optimized during assembly in the endoplasmic reticulum (ER) and depends on the collective function of components of the peptide-loading complex (PLC). The chaperone-like molecule tapasin is the cornerstone of this complex and acts directly on the MHC class I molecule to promote high-affinity peptide loading. Optimal tapasin activity, however, relies on the ability of ERp57 and calreticulin, two proteins involved in general ER glycoprotein folding, to bridge and thereby stabilize its otherwise weak interaction with the MHC class I heavy chain. Here, we describe methods for the recombinant expression of soluble components of the PLC specifically tailored to generate the post-translational modifications required to support subcomplex assembly in vitro. Using recombinant MHC class I molecules bearing monoglucosylated N-linked glycans, calreticulin, and disulfide-linked tapasin/ERp57 heterodimers, this soluble PLC subcomplex can be employed to study the mechanism of peptide loading or the principles governing peptide selection for particular MHC class I alleles.

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Acknowledgement

This work was supported by the Howard Hughes Medical Institute (P.C.).

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Authors and Affiliations

  1. Department of Pathology, Case Western Reserve University, Cleveland, OH, USA

    Pamela A. Wearsch

  2. Department of Immunobiology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, USA

    Peter Cresswell

Authors
  1. Pamela A. Wearsch
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  2. Peter Cresswell
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Corresponding author

Correspondence to Peter Cresswell .

Editor information

Editors and Affiliations

  1. Institut National de la Santé et de la Recherche Médicale, Unité 1013, Université Paris Descartes, Faculté de médecine, Paris, France

    Peter van Endert

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Wearsch, P.A., Cresswell, P. (2013). In Vitro Reconstitution of the MHC Class I Peptide-Loading Complex. In: van Endert, P. (eds) Antigen Processing. Methods in Molecular Biology™, vol 960. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-218-6_6

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  • DOI: https://doi.org/10.1007/978-1-62703-218-6_6

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-217-9

  • Online ISBN: 978-1-62703-218-6

  • eBook Packages: Springer Protocols

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