Recombinant single chain human HLA-DR1 molecules as biologically active covalently linked heterotrimers of antigenic peptide, beta chain, and alpha chain

Xiaojie Zhu, Scheheraza Sadegh-Nasseri, Sina Bavari, Robert G. Ulrich, Michael Mage

Research output: Contribution to journalArticle

Abstract

We engineered HLA-DR1 cDNA to encode influenza HA or HIV gag peptides covalently attached via a peptide linker to the amino terminus of the DR1 beta chain. Cotransfection with DR alpha cDNA into mouse L cells resulted in surface expression of HLA-DR1 molecules that reacted with mAbs specific for correctly folded HLA-DR epitopes. To ease production of these molecules, we engineered an additional peptide linker between the carboxyl terminus of a truncated beta chain (without TM or cytoplasmic domains) and the amino terminus of full length DR alpha chain. On transfection, the entire heterotrimer of peptide, beta chain, and alpha chain was expressed as a single covalently linked polypeptide chain with an expected molecular mass of approximately 66 kDa. Affinity purified soluble secreted single chain molecules (with truncated alpha chain) electrophorese as expected for compact Class II MHC dimers. Cell surface two-chain or single chain HLA-DR1 molecules with a covalent HA peptide can bind the SEA and SEB superantigens. They can also stimulate HLA-DR1 restricted HA-specific T cells. We plan to analyse the folding pathways of these recombinant MHC molecules and to study their in vitro immunogenicity.

Original languageEnglish (US)
JournalFASEB Journal
Volume10
Issue number6
StatePublished - 1996
Externally publishedYes

Fingerprint

HLA-DR1 Antigen
peptides
Peptides
Molecules
superantigens
Complementary DNA
transfection
T-cells
influenza
in vitro studies
epitopes
Molecular mass
HLA-DR Antigens
polypeptides
Dimers
T-lymphocytes
Human Influenza
Transfection
immune response
Epitopes

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Recombinant single chain human HLA-DR1 molecules as biologically active covalently linked heterotrimers of antigenic peptide, beta chain, and alpha chain. / Zhu, Xiaojie; Sadegh-Nasseri, Scheheraza; Bavari, Sina; Ulrich, Robert G.; Mage, Michael.

In: FASEB Journal, Vol. 10, No. 6, 1996.

Research output: Contribution to journalArticle

@article{655243c097334c719c1b63256c83ab67,
title = "Recombinant single chain human HLA-DR1 molecules as biologically active covalently linked heterotrimers of antigenic peptide, beta chain, and alpha chain",
abstract = "We engineered HLA-DR1 cDNA to encode influenza HA or HIV gag peptides covalently attached via a peptide linker to the amino terminus of the DR1 beta chain. Cotransfection with DR alpha cDNA into mouse L cells resulted in surface expression of HLA-DR1 molecules that reacted with mAbs specific for correctly folded HLA-DR epitopes. To ease production of these molecules, we engineered an additional peptide linker between the carboxyl terminus of a truncated beta chain (without TM or cytoplasmic domains) and the amino terminus of full length DR alpha chain. On transfection, the entire heterotrimer of peptide, beta chain, and alpha chain was expressed as a single covalently linked polypeptide chain with an expected molecular mass of approximately 66 kDa. Affinity purified soluble secreted single chain molecules (with truncated alpha chain) electrophorese as expected for compact Class II MHC dimers. Cell surface two-chain or single chain HLA-DR1 molecules with a covalent HA peptide can bind the SEA and SEB superantigens. They can also stimulate HLA-DR1 restricted HA-specific T cells. We plan to analyse the folding pathways of these recombinant MHC molecules and to study their in vitro immunogenicity.",
author = "Xiaojie Zhu and Scheheraza Sadegh-Nasseri and Sina Bavari and Ulrich, {Robert G.} and Michael Mage",
year = "1996",
language = "English (US)",
volume = "10",
journal = "FASEB Journal",
issn = "0892-6638",
publisher = "FASEB",
number = "6",

}

TY - JOUR

T1 - Recombinant single chain human HLA-DR1 molecules as biologically active covalently linked heterotrimers of antigenic peptide, beta chain, and alpha chain

AU - Zhu, Xiaojie

AU - Sadegh-Nasseri, Scheheraza

AU - Bavari, Sina

AU - Ulrich, Robert G.

AU - Mage, Michael

PY - 1996

Y1 - 1996

N2 - We engineered HLA-DR1 cDNA to encode influenza HA or HIV gag peptides covalently attached via a peptide linker to the amino terminus of the DR1 beta chain. Cotransfection with DR alpha cDNA into mouse L cells resulted in surface expression of HLA-DR1 molecules that reacted with mAbs specific for correctly folded HLA-DR epitopes. To ease production of these molecules, we engineered an additional peptide linker between the carboxyl terminus of a truncated beta chain (without TM or cytoplasmic domains) and the amino terminus of full length DR alpha chain. On transfection, the entire heterotrimer of peptide, beta chain, and alpha chain was expressed as a single covalently linked polypeptide chain with an expected molecular mass of approximately 66 kDa. Affinity purified soluble secreted single chain molecules (with truncated alpha chain) electrophorese as expected for compact Class II MHC dimers. Cell surface two-chain or single chain HLA-DR1 molecules with a covalent HA peptide can bind the SEA and SEB superantigens. They can also stimulate HLA-DR1 restricted HA-specific T cells. We plan to analyse the folding pathways of these recombinant MHC molecules and to study their in vitro immunogenicity.

AB - We engineered HLA-DR1 cDNA to encode influenza HA or HIV gag peptides covalently attached via a peptide linker to the amino terminus of the DR1 beta chain. Cotransfection with DR alpha cDNA into mouse L cells resulted in surface expression of HLA-DR1 molecules that reacted with mAbs specific for correctly folded HLA-DR epitopes. To ease production of these molecules, we engineered an additional peptide linker between the carboxyl terminus of a truncated beta chain (without TM or cytoplasmic domains) and the amino terminus of full length DR alpha chain. On transfection, the entire heterotrimer of peptide, beta chain, and alpha chain was expressed as a single covalently linked polypeptide chain with an expected molecular mass of approximately 66 kDa. Affinity purified soluble secreted single chain molecules (with truncated alpha chain) electrophorese as expected for compact Class II MHC dimers. Cell surface two-chain or single chain HLA-DR1 molecules with a covalent HA peptide can bind the SEA and SEB superantigens. They can also stimulate HLA-DR1 restricted HA-specific T cells. We plan to analyse the folding pathways of these recombinant MHC molecules and to study their in vitro immunogenicity.

UR - http://www.scopus.com/inward/record.url?scp=33749082931&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33749082931&partnerID=8YFLogxK

M3 - Article

AN - SCOPUS:33749082931

VL - 10

JO - FASEB Journal

JF - FASEB Journal

SN - 0892-6638

IS - 6

ER -