| 摘要 |
FIELD: medicine. ^ SUBSTANCE: described is recombinant plasmid DNA pET-32a/TRII. Plasmid consists of BgHI/Hindlll-fragment of plasmid pET-32a DNA, containing lacl-promoter E.coli, T7 promoter, Trx-Tag coding sequence, His-Tag coding sequence, S-Tag coding sequence, T7 terminator, lad-coding sequence, ba-coding sequence, sequence fl site ori of replication initiation, as well as Bg1II/Hindlll gene fragment, coding DNA of synthetic gene TRII. Described is strain of bacteria Escherichia coli BL21 (DE3)/pET-32a/TRII - producent of fusion protein thioredoxin- TRII. Claimed is method of isolating target protein of human TRII. Soluble fraction of cytoplasmatic proteins is isolated from strain-producent Escherichia coli BL21(DE3)/pET-32a/TRII. Chromatographic purification with further renaturation of fusion protein thioredoxin- TRII is carried out. After that, protein is incubated for 72 hours at 4C in buffer, containing 0.5 M of arginine, in presence of oxidised and reduced glutathione in ratio 1:50 mM respectively. After that dialysis, decomposition of catalytic sununit with recombinant enteropeptidase and purification of target protein TRII from thioredoxin are carried out. ^ EFFECT: invention makes it possible to obtain increased output of highly purified human TRII protein with native N-end. ^ 3 cl, 3 dwg, 1 tbl, 3 ex |
