Reassembly and reconstitution of separate alpha and beta chains of human leukocyte antigen DR4 molecule isolated from Escherichia coli.
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 alpha- and beta-chains. In the present study, we cloned and expressed the alpha- and beta-chain of HLA-DR4 lacking the transmembrane and cytoplasmic domain separately in Escherichia coli using the pET-5a expression vector system. The expressed alpha- and beta-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-alpha 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.
Kang
J H
JH
Peptide Engineering Research Unit, Korea Research Institute of Bioscience and Biotechnology, KIST, Taejon, Korea.
Maeng
C Y
CY
Park
J H
JH
Hahm
K S
KS
Han
B D
BD
Kim
K L
KL
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015804:HLA-DR4 Antigen / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D015804:HLA-DR4 Antigen; D011994:Recombinant Proteins