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Title 

Reassembly and reconstitution of separate αand βchains of human leukocyte antigen DR4 molecule isolated from Escherichia coli

Authors 

Joo Hyun KangCheol-Young MaengJung Hyun ParkKyung Soo HahmByoung Don HanKil Lyong Kim

Publisher 

Springer Verlag (Germany)

Issue Date 

1997

Citation 

Molecules and Cells, vol. 7, no. 2, pp. 237-243

Keywords 

HLA DR4 antigenEscherichia colihumanisolation and purification

Abstract 

The class II major histocompatibility complex molecules play a major role in presentation of exogenous antigenic peptides to the CD4 positive helper T cell. These are heterodimeric cell surface glycoproteins consisting of α- and β-chains. In the present study, we cloned and expressed the α- and β-chain of HLA-DR4 lacking the transmembrane and cytoplasmic domain separately in Escherichia coli using the pET-5a expression vector system. The expressed α-and β-chains were purified in a denaturing condition by an ion exchange chromatography on Q-Sepharose and a gel filtration chromatography on Sephacryl S-200, respectively. The recombinant proteins were refolded and reassembled by removing the denaturing agent and concomitant reoxidation of the disulfide bond. The refolded heterodimeric rDR4 molecule was resolved by 12.5% SDS-PAGE in a nonreducing condition and confirmed by Western blot using polyclonal antibody against DR-α and the monoclonal antibody (L243) for the conformationally correct DR molecule. The rDR4 molecules were reconstituted with a 50-fold molar excess biot-HA (307-319), and the bound peptides to the heterodimer complex were determined by a microplate assay coated with L243 antibody using Extravidin-HRP conjugate.

ISSN 

1016-8478

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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