Empty and Peptide-Loaded Class II Major Histocompatibility Complex Proteins Produced by Expression inEscherichia coliand Foldingin Vitro

Frayser, Mia; Sato, Aaron K.; Xu, Lihui; Stern, Lawrence J.

doi:10.1006/prep.1998.0987

Cited by 95 publications

(120 citation statements)

References 49 publications

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“…In both cases, the (R ) 2 dimer (Figure 2, dashed line) and higher-order oligomers could be excluded. The extracted values for the molecular masses of empty DR1 (45.0 ( 2.9 kDa) and DR1-Ha (47.0 ( 3.0 kDa) are close to the calculated monomeric values of 43.2 kDa for the nonhydrated empty protein and 44.6 kDa for the Ha peptide complex (31).…”

Section: Resultssupporting

confidence: 78%

“…We observed differences in thermodynamic parameters between the empty and peptide-loaded DR1, as measured by thermal denaturation experiments. MHC thermal denaturation is in general irreversible, in that completely denatured protein will not spontaneously refold upon cooling (25)(26)(27)(28)(29)31). However, in our experiments, the melting temperature and extracted thermodynamic values were essentially independent of scan rate, indicating that these values reflect the underlying reversible folding transition and not a subsequent irreversible denaturation step (40).…”

Section: Resultsmentioning

confidence: 59%

“…We used a soluble recombinant form of the protein produced by expressing DR1 R-and -subunits separately in E. coli inclusion bodies and subsequently folding the subunits together in vitro (31). The folded material is free of contaminating proteins and peptides and is fully active in binding antigenic peptides ( Figure 1A), as indicated by complete resistance to SDS-induced chain dissociation after peptide loading (DR1-Ha, Figure 1B).…”

Section: Resultsmentioning

confidence: 99%

“…In addition, empty class II MHC proteins produced in recombinant systems have exhibited a pronounced tendency to aggregate (30), and the presence of these aggregates complicates physical analysis of the protein. In this study, we used homogeneous, monomeric, soluble forms of empty and peptide-loaded HLA-DR1 complexes produced by expression in Escherichia coli and folding in vitro (31).…”

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confidence: 99%

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A Conformational Change in the Human Major Histocompatibility Complex Protein HLA-DR1 Induced by Peptide Binding

et al. 1999

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To investigate a conformational change accompanying peptide binding to class II MHC proteins, we probed the structure of a soluble version of the human class II MHC protein HLA-DR1 in empty and peptide-loaded forms. Peptide binding induced a large decrease in the effective radius of the protein as determined by gel filtration, dynamic light scattering, and analytical ultracentrifugation. It caused a substantial increase in the cooperativity of thermal denaturation and induced alterations in MHC polypeptide backbone structure as determined by circular dichroism. These changes suggest a condensation of the protein around the bound peptide. An antibody specific for 58-69 preferentially bound the empty protein, indicating that the peptide-induced conformational change involves the -subunit helical region. The conformational change may have important implications for the mechanisms of intracellular antigen presentation pathways.

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Section: Resultssupporting

confidence: 78%

Section: Resultsmentioning

confidence: 59%

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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A Conformational Change in the Human Major Histocompatibility Complex Protein HLA-DR1 Induced by Peptide Binding

et al. 1999

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“…The soluble human class II MHC HLA-DR1 was produced in E. coli as separate inclusion bodies for the extracellular domains of the ␣-and ␤-chains and folded in vitro by rapid dilution in the presence of excess peptide as previously described (28). In some proteins, a uniquely reactive cysteine was introduced at the C terminus of either the ␣ or ␤ extracellular domain for the ability to cross-link into oligomers.…”

Section: Production Labeling and Cross-linking Of Soluble Class II mentioning

confidence: 99%

CD8 T Cells, Like CD4 T Cells, Are Triggered by Multivalent Engagement of TCRs by MHC-Peptide Ligands but Not by Monovalent Engagement

Stone

Stern

2006

The Journal of Immunology

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T cell activation is initiated by recognition of antigenic peptide presented in complex with MHC molecules on the surface of APCs. The mechanism by which this recognition occurs is still unclear, and many models exist in the literature. CD4 T cells have been shown to respond to soluble oligomers of activating class II MHC-peptide complexes, but not to soluble monomers. In determining the reactivity of CD8 T cells to soluble activating class I MHC-peptide complexes, a complicating phenomenon had been observed whereby peptide from soluble complexes was loaded onto cell surface MHCs on the T cells and re-presented to other T cells, clouding the true valency requirement for activation. This study uses soluble allogeneic class I MHC-peptide monomers and oligomers to stimulate murine CD8 T cells without the possible complication of peptide re-presentation. The results show that MHC class I monomers bind to, but do not activate, CD8 T cells whether the cells are in solution or adhered to a surface. Monomeric MHC class I binding can antagonize the stimulation triggered by soluble oligomers, a phenomenon also observed for CD4 T cells. Dimeric engagement is necessary and sufficient to stimulate downstream activation processes including TCR down-regulation, Zap70 phosphorylation, and CD25 and CD69 up-regulation, even in T cells that do not express the MHC coreceptor CD8. Thus, the valency dependence of the response of CD8 T cells to soluble MHC-peptide reagents is the same as previously observed for CD4 T cells.

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Measurement of Peptide Binding to MHC Class II Molecules by Fluorescence Polarization

Yin

Stern

2014

CP in Immunology

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Peptide binding to major histocompatibility complex class II (MHCII) molecules is a key process in antigen presentation and CD4+ T cell epitope selection. This unit describes a fairly simple but powerful fluorescence polarization-based binding competition assay to measure peptide binding to soluble recombinant MHCII molecules. The binding of a peptide of interest to MHCII molecules is assessed based on its ability to inhibit the binding of a fluorescence-labeled probe peptide, with the strength of binding characterized as IC50 (concentration required for 50% inhibition of probe peptide binding). Data analysis related to this method is discussed. In addition, this unit includes a support protocol for fluorescence labeling peptide using an amine-reactive probe. The advantage of this protocol is that it allows simple, fast, and high throughput measurements of binding for a large set of peptides to MHCII molecules.

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Empty and Peptide-Loaded Class II Major Histocompatibility Complex Proteins Produced by Expression inEscherichia coliand Foldingin Vitro

Cited by 95 publications

References 49 publications

A Conformational Change in the Human Major Histocompatibility Complex Protein HLA-DR1 Induced by Peptide Binding

A Conformational Change in the Human Major Histocompatibility Complex Protein HLA-DR1 Induced by Peptide Binding

CD8 T Cells, Like CD4 T Cells, Are Triggered by Multivalent Engagement of TCRs by MHC-Peptide Ligands but Not by Monovalent Engagement

Measurement of Peptide Binding to MHC Class II Molecules by Fluorescence Polarization

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