Formation of functional peptide complexes of class II major histocompatibility complex proteins from subunits produced in Escherichia coli.

Abstract

Class H major histocompatibility complex molecules play a major role in the iimmune response by binding peptide fragments of exogenous antigens and displaying them on the surfaces of antigen-presenting ceils, where they can be recognized by T ceils. To facilitate structural and functional studies of these molecules, we have produced truncated a and 13 chains of the murine class HI molecule I-Ek in Escherichia coli (Ec-I-Ei) and have developed conditions to fold them in the presence of specific peptides with yields of complex approaching 2%. Reconstitution is specific since only unlabeled peptides known to bind I-Ek compete with biotinylated peptide, as asese by ELISA. Complexes of the refolded heterodimer (Ec-I-Ek) with either of two different peptide antigens remain associted during nonreducing SDS/PAGE. Immobilized Ec-I-E"-.peptide complexes stimulate Iymphokine production by three T-cell clones in an antigen-specific manner with a doseresponse relation comparable to previously described soluble I-Ek molecules produced in CHO cells. These results demonstrate that folding of e. and Ep polypeptides does not require any other protein to produce the biologically relevant conformation and that carbohydrate modification of this class H. molecule is not necessary for a13 T-cell recognition.

DM Loss in k Haplotype Mice Reveals Isotype-Specific Chaperone Requirements

“…Perhaps the intrinsic stability of these empty dimers differs, and E k displays enhanced peptide-loading capabilities due to its extended half-life inside endocytic compartments. Peptide binding by empty E k has been extensively assessed in vitro (54,(75)(76)(77)(78)(79), but less is known about E d conformational changes during peptide capture. A buried cluster of acidic residues surrounding the P6 pocket is conserved in all mouse I-E and human DR molecules, and it seems likely that these contacts are available only inside endocytic compartments (80).…”

Quantitation of CD8+T-Lymphocyte Responses to Multiple Epitopes from Simian Virus 40 (SV40) Large T Antigen in C57BL/6 Mice Immunized with SV40, SV40 T-Antigen-Transformed Cells, or Vaccinia Virus Recombinants Expressing Full-Length T Antigen or Epitope Minigenes

“…Plasmids encoding ␤ 2 -microglobulin (␤ 2 m) were pET3␣-bo␤2m (bovine ␤ 2 m) and pET3␣-hu␤2m (both kindly provided by D. Shields and R. Ribaudo, then of the National Institute of Allergy and Infectious Diseases). All plasmids were separately introduced into Escherichia coli strain BL21 (DE3), and subunit production was induced with 1 mM isopropyl-␤-D-thiogalactopyranoside as described previously (4).…”

Tetramers reveal IL-17–secreting CD4+T cells that are specific for U1-70 in lupus and mixed connective tissue disease

“…Production of I-Ek Tetramers and Peptide Exchange. The expression and purification of recombinant I-E k monomers have been described previously (50). In brief, I-E k α and β chains were expressed in Escherichia coli as inclusion bodies, refolded, and purified with a conditional peptide ligand (an N-terminal His6-tagged MCC peptide with K99ANP; see above).…”