A single-chain fusion molecule consisting of peptide, major histocompatibility gene complex class I heavy chain and beta2-microglobulin can fold partially correctly, but binds peptide inefficiently

C Sylvester-Hvid; S Buus

A single-chain fusion molecule consisting of peptide, major histocompatibility gene complex class I heavy chain and beta2-microglobulin can fold partially correctly, but binds peptide inefficiently

C Sylvester-Hvid, S Buus

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

6 Citationer (Scopus)

Abstract

Udgivelsesdato: 1999-Oct

Originalsprog	Engelsk
Tidsskrift	Scandinavian Journal of Immunology
Vol/bind	50
Udgave nummer	4
Sider (fra-til)	355-62
Antal sider	7
ISSN	0300-9475
Status	Udgivet - 1999

Bibliografisk note

Keywords: Antigen Presentation; Hemagglutinin Glycoproteins, Influenza Virus; Histocompatibility Antigens Class I; Humans; Models, Immunological; Nickel; Nitrilotriacetic Acid; Peptide Fragments; Protein Binding; Protein Engineering; Protein Folding; Receptors, Antigen, T-Cell; Recombinant Fusion Proteins; Sepharose; beta 2-Microglobulin

Citationsformater

A single-chain fusion molecule consisting of peptide, major histocompatibility gene complex class I heavy chain and beta2-microglobulin can fold partially correctly, but binds peptide inefficiently. / Sylvester-Hvid, C ; Buus, S.

I: Scandinavian Journal of Immunology, Bind 50, Nr. 4, 1999, s. 355-62.

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

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title = "A single-chain fusion molecule consisting of peptide, major histocompatibility gene complex class I heavy chain and beta2-microglobulin can fold partially correctly, but binds peptide inefficiently",

abstract = "The function of major histocompatibility complex class I (MHC-I) molecules is to sample peptides from the intracellular environment and present these peptides to CD8+ cytotoxic T lymphocytes (CTL). We have attempted to develop a general approach to produce large amounts of pure and active recombinant MHC-I molecules. A convenient source of MHC-I molecules would be a valuable tool in structural and biochemical analysis of MHC-I, and in experiments using MHC-I molecules to enable specific manipulations of experimental and physiological CTL responses. Here we describe the generation of a recombinant murine MHC-I molecule, which could be produced in large amounts in bacteria. The recombinant MHC-I protein was expressed as a single molecule (PepSc) consisting of the antigenic peptide linked to the MHC-I heavy chain and further linked to human beta2-microglobulin (hbeta2m). The PepSc molecule was denatured, extracted, purified and folded using a recently developed in vitro reiterative refolding strategy. This led to the formation of soluble, recombinant MHC-I molecules, which migrated as monomers of the expected size when submitted to non-reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Serological analysis revealed the presence of some, but not all, MHC-I-specific epitopes. Biochemically, PepSc could bind peptide, however, rather ineffectively. We suggest that a partially correctly refolded MHC-I has been obtained.",

author = "C Sylvester-Hvid and S Buus",

note = "Keywords: Antigen Presentation; Hemagglutinin Glycoproteins, Influenza Virus; Histocompatibility Antigens Class I; Humans; Models, Immunological; Nickel; Nitrilotriacetic Acid; Peptide Fragments; Protein Binding; Protein Engineering; Protein Folding; Receptors, Antigen, T-Cell; Recombinant Fusion Proteins; Sepharose; beta 2-Microglobulin",

year = "1999",

language = "English",

volume = "50",

pages = "355--62",

journal = "Scandinavian Journal of Immunology, Supplement",

issn = "0301-6323",

publisher = "Wiley-Blackwell",

number = "4",

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TY - JOUR

T1 - A single-chain fusion molecule consisting of peptide, major histocompatibility gene complex class I heavy chain and beta2-microglobulin can fold partially correctly, but binds peptide inefficiently

AU - Sylvester-Hvid, C

AU - Buus, S

N1 - Keywords: Antigen Presentation; Hemagglutinin Glycoproteins, Influenza Virus; Histocompatibility Antigens Class I; Humans; Models, Immunological; Nickel; Nitrilotriacetic Acid; Peptide Fragments; Protein Binding; Protein Engineering; Protein Folding; Receptors, Antigen, T-Cell; Recombinant Fusion Proteins; Sepharose; beta 2-Microglobulin

PY - 1999

Y1 - 1999

N2 - The function of major histocompatibility complex class I (MHC-I) molecules is to sample peptides from the intracellular environment and present these peptides to CD8+ cytotoxic T lymphocytes (CTL). We have attempted to develop a general approach to produce large amounts of pure and active recombinant MHC-I molecules. A convenient source of MHC-I molecules would be a valuable tool in structural and biochemical analysis of MHC-I, and in experiments using MHC-I molecules to enable specific manipulations of experimental and physiological CTL responses. Here we describe the generation of a recombinant murine MHC-I molecule, which could be produced in large amounts in bacteria. The recombinant MHC-I protein was expressed as a single molecule (PepSc) consisting of the antigenic peptide linked to the MHC-I heavy chain and further linked to human beta2-microglobulin (hbeta2m). The PepSc molecule was denatured, extracted, purified and folded using a recently developed in vitro reiterative refolding strategy. This led to the formation of soluble, recombinant MHC-I molecules, which migrated as monomers of the expected size when submitted to non-reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Serological analysis revealed the presence of some, but not all, MHC-I-specific epitopes. Biochemically, PepSc could bind peptide, however, rather ineffectively. We suggest that a partially correctly refolded MHC-I has been obtained.

AB - The function of major histocompatibility complex class I (MHC-I) molecules is to sample peptides from the intracellular environment and present these peptides to CD8+ cytotoxic T lymphocytes (CTL). We have attempted to develop a general approach to produce large amounts of pure and active recombinant MHC-I molecules. A convenient source of MHC-I molecules would be a valuable tool in structural and biochemical analysis of MHC-I, and in experiments using MHC-I molecules to enable specific manipulations of experimental and physiological CTL responses. Here we describe the generation of a recombinant murine MHC-I molecule, which could be produced in large amounts in bacteria. The recombinant MHC-I protein was expressed as a single molecule (PepSc) consisting of the antigenic peptide linked to the MHC-I heavy chain and further linked to human beta2-microglobulin (hbeta2m). The PepSc molecule was denatured, extracted, purified and folded using a recently developed in vitro reiterative refolding strategy. This led to the formation of soluble, recombinant MHC-I molecules, which migrated as monomers of the expected size when submitted to non-reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Serological analysis revealed the presence of some, but not all, MHC-I-specific epitopes. Biochemically, PepSc could bind peptide, however, rather ineffectively. We suggest that a partially correctly refolded MHC-I has been obtained.

M3 - Journal article

C2 - 10520174

VL - 50

SP - 355

EP - 362

JO - Scandinavian Journal of Immunology, Supplement

JF - Scandinavian Journal of Immunology, Supplement

SN - 0301-6323

IS - 4

ER -