| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2005: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2004: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
In the present study, the extracellular domains of a human γδ T cell receptor were expressed in E.coli, refolded together, and crystallized, then the crystal structure was determined at a 2.2Å resolution. In practice, cDNAs encoding the extracellular domains of TCR γ- and δ-chains were separately cloned and incorporated into a T7-based expression vector. Since E.coli transfected with the original cDNA/vector failed to produce the recombinant proteins, the predicted step loop structure was removed on the basis of molecular dynamics caliculation. In addition, several codons were replaced by E.coli-favored ones and the E.coli codon index was increased up to 0.65. After the gene modification, the recombinant proteins could be produced in E.coli at 100 mg/l culture or more. The inclusion bodies were dissolved in guanidine solution, treated with DTT and refolded in an arginine-based buffer using a rapid dilution method. After dialysis, the protein sample was brought to pH 5.5 judging from pl and purified on cation-exchange column chromatography, followed by gel filtration. In screening for crystallization, commercial and in-house made kits were used. After 1 week, crystals appeared in several conditions containing PEG4000. After improvement of the conditions, the crystals were analyzed by the in-house X-ray generator, which gave a 3.0Å resolution. Then, the crystals were further analyzed by SPring 8. The data were processed on workstation and the following statistical values were obtained : space group ; P21, unit ; 60.82, 77.35, 100.12, 90.00, 95.00, and 90.00, reflection ; 314, 713, independent reflection ; 65, 401, resolution ; 1.9, completeness ; 91.4, redundancy ; 4.8, and refinement ; 15-1.90. As described above, crystal structure of human γδ T cell receptor was solved at a high resolution
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