Functional recombinant MHC class II molecules and high-throughput peptide-binding assays

Sune Justesen; Mikkel Harndahl; Kasper Lamberth; Lise-Lotte B Nielsen; Søren Buus

doi:10.1186/1745-7580-5-2

Functional recombinant MHC class II molecules and high-throughput peptide-binding assays

Sune Justesen, Mikkel Harndahl, Kasper Lamberth, Lise-Lotte B Nielsen, Søren Buus

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

53 Citationer (Scopus)

Abstract

Udgivelsesdato: 2009

Originalsprog	Engelsk
Tidsskrift	Immunome Research
Vol/bind	5
Udgave nummer	2
ISSN	1745-7580
DOI	https://doi.org/10.1186/1745-7580-5-2
Status	Udgivet - 2009

Adgang til dokumentet

10.1186/1745-7580-5-2

Citationsformater

@article{138c4b60779811df928f000ea68e967b,

title = "Functional recombinant MHC class II molecules and high-throughput peptide-binding assays",

abstract = "BACKGROUND: Molecules of the class II major histocompability complex (MHC-II) specifically bind and present exogenously derived peptide epitopes to CD4+ T helper cells. The extreme polymorphism of the MHC-II hampers the complete analysis of peptide binding. It is also a significant hurdle in the generation of MHC-II molecules as reagents to study and manipulate specific T helper cell responses. Methods to generate functional MHC-II molecules recombinantly, and measure their interaction with peptides, would be highly desirable; however, no consensus methodology has yet emerged. RESULTS: We generated alpha and beta MHC-II chain constructs, where the membrane-spanning regions were replaced by dimerization motifs, and the C-terminal of the beta chains was fused to a biotinylation signal peptide (BSP) allowing for in vivo biotinylation. These chains were produced separately as inclusion bodies in E. coli , extracted into urea, and purified under denaturing and non-reducing conditions using conventional column chromatography. Subsequently, diluting the two chains into a folding reaction with appropriate peptide resulted in efficient peptide-MHC-II complex formation. Several different formats of peptide-binding assay were developed including a homogeneous, non-radioactive, high-throughput (HTS) binding assay. Binding isotherms were generated allowing the affinities of interaction to be determined. The affinities of the best binders were found to be in the low nanomolar range. Recombinant MHC-II molecules and accompanying HTS peptide-binding assay were successfully developed for nine different MHC-II molecules including the DPA1*0103/DPB1*0401 (DP401) and DQA1*0501/DQB1*0201, where both alpha and beta chains are polymorphic, illustrating the advantages of producing the two chains separately. CONCLUSION: We have successfully developed versatile MHC-II resources, which may assist in the generation of MHC class II -wide reagents, data, and tools.",

author = "Sune Justesen and Mikkel Harndahl and Kasper Lamberth and Nielsen, {Lise-Lotte B} and S{\o}ren Buus",

year = "2009",

doi = "10.1186/1745-7580-5-2",

language = "English",

volume = "5",

journal = "Immunome Research",

issn = "1745-7580",

publisher = "BioMed Central Ltd.",

number = "2",

}

TY - JOUR

T1 - Functional recombinant MHC class II molecules and high-throughput peptide-binding assays

AU - Justesen, Sune

AU - Harndahl, Mikkel

AU - Lamberth, Kasper

AU - Nielsen, Lise-Lotte B

AU - Buus, Søren

PY - 2009

Y1 - 2009

N2 - BACKGROUND: Molecules of the class II major histocompability complex (MHC-II) specifically bind and present exogenously derived peptide epitopes to CD4+ T helper cells. The extreme polymorphism of the MHC-II hampers the complete analysis of peptide binding. It is also a significant hurdle in the generation of MHC-II molecules as reagents to study and manipulate specific T helper cell responses. Methods to generate functional MHC-II molecules recombinantly, and measure their interaction with peptides, would be highly desirable; however, no consensus methodology has yet emerged. RESULTS: We generated alpha and beta MHC-II chain constructs, where the membrane-spanning regions were replaced by dimerization motifs, and the C-terminal of the beta chains was fused to a biotinylation signal peptide (BSP) allowing for in vivo biotinylation. These chains were produced separately as inclusion bodies in E. coli , extracted into urea, and purified under denaturing and non-reducing conditions using conventional column chromatography. Subsequently, diluting the two chains into a folding reaction with appropriate peptide resulted in efficient peptide-MHC-II complex formation. Several different formats of peptide-binding assay were developed including a homogeneous, non-radioactive, high-throughput (HTS) binding assay. Binding isotherms were generated allowing the affinities of interaction to be determined. The affinities of the best binders were found to be in the low nanomolar range. Recombinant MHC-II molecules and accompanying HTS peptide-binding assay were successfully developed for nine different MHC-II molecules including the DPA1*0103/DPB1*0401 (DP401) and DQA1*0501/DQB1*0201, where both alpha and beta chains are polymorphic, illustrating the advantages of producing the two chains separately. CONCLUSION: We have successfully developed versatile MHC-II resources, which may assist in the generation of MHC class II -wide reagents, data, and tools.

AB - BACKGROUND: Molecules of the class II major histocompability complex (MHC-II) specifically bind and present exogenously derived peptide epitopes to CD4+ T helper cells. The extreme polymorphism of the MHC-II hampers the complete analysis of peptide binding. It is also a significant hurdle in the generation of MHC-II molecules as reagents to study and manipulate specific T helper cell responses. Methods to generate functional MHC-II molecules recombinantly, and measure their interaction with peptides, would be highly desirable; however, no consensus methodology has yet emerged. RESULTS: We generated alpha and beta MHC-II chain constructs, where the membrane-spanning regions were replaced by dimerization motifs, and the C-terminal of the beta chains was fused to a biotinylation signal peptide (BSP) allowing for in vivo biotinylation. These chains were produced separately as inclusion bodies in E. coli , extracted into urea, and purified under denaturing and non-reducing conditions using conventional column chromatography. Subsequently, diluting the two chains into a folding reaction with appropriate peptide resulted in efficient peptide-MHC-II complex formation. Several different formats of peptide-binding assay were developed including a homogeneous, non-radioactive, high-throughput (HTS) binding assay. Binding isotherms were generated allowing the affinities of interaction to be determined. The affinities of the best binders were found to be in the low nanomolar range. Recombinant MHC-II molecules and accompanying HTS peptide-binding assay were successfully developed for nine different MHC-II molecules including the DPA1*0103/DPB1*0401 (DP401) and DQA1*0501/DQB1*0201, where both alpha and beta chains are polymorphic, illustrating the advantages of producing the two chains separately. CONCLUSION: We have successfully developed versatile MHC-II resources, which may assist in the generation of MHC class II -wide reagents, data, and tools.

U2 - 10.1186/1745-7580-5-2

DO - 10.1186/1745-7580-5-2

M3 - Journal article

C2 - 19416502

SN - 1745-7580

VL - 5

JO - Immunome Research

JF - Immunome Research

IS - 2

ER -