The production, purification and crystallization of a soluble heterodimeric form of a highly selected T-cell receptor in its unliganded and liganded state.
T-cell antigen receptors (TcRs) are heterodimeric cell-surface receptors that play a pivotal role in the cellular immune response. The TcR interacts specifically with a peptide-laden major histocompatability complex (pMHC). A human TcR has been characterized that interacts with an immunodominant epitope, FLRGRAYGL, from the Epstein-Barr virus, a ubiquitous human pathogen, in complex with HLA-B8. Despite the vast TcR repertoire, this TcR is found in up to 10% of the total T-cell population in seropositive HLA-B8+ individuals. In this report, this highly selected TcR is characterized by expressing in Escherichia coli, refolding, purifying and crystallizing the receptor. In addition, the HLA-B8-FLRGRAYGL complex has been expressed in E. coli, refolded and shown to be functionally active. Using native gel electrophoresis, the refolded TcR is shown to be capable of binding specifically to the refolded HLA-B8-FLRGRAYGL and this TcR has been crystallized in complex with the pMHC. The crystals of the unliganded and liganded TcR diffract to 1.5 and 2.5 A, respectively.
Clements
Craig S
CS
The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3168, Australia.
Kjer-Nielsen
Lars
L
MacDonald
Whitney A
WA
Brooks
Andrew G
AG
Purcell
Anthony W
AW
McCluskey
James
J
Rossjohn
Jamie
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D003460:Crystallization; D017931:DNA Primers; D019281:Dimerization; D008024:Ligands; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D008024:Ligands; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins