Recombinant plasmid DNA pTEV-TMS encoding a fusion protein intended for proteolytic cleavage with the formation of the antiangiogenic peptide of the tumumin-derived fragment L69K-95 of human tumstatin, strain E. coli BL21 (DE3) / pTEV-TMS-producer of the specified protein and method for producing it recombinant antiangiogenic peptide

摘要

1. Recombinant plasmid DNA pTEV-TMS, encoding the TrxTEVrs-TMS fusion protein containing thioredoxin and the Tumastin antiangiogenic peptide (TMS), which is a modified fragment [L69K-95] of human tumstatin with Ser- sequences attached to the N-terminal and C-ends Gly-Ala-Met-Gly and Pro-Gly-Pro, respectively, separated by a site for cleavage by the proteinase of the tobacco engraving virus (TEV proteinase), consisting of XbaI / BamHI, a fragment of the plasmid vector pTEV containing the replication initiation site of the plasmid vector (Ori) and phage M13 (M13 ori), gene repre lactose operon sora (lacI), T7 bacteriophage RNA polymerase transcription promoter and terminator, RNA organizing protein gene (Rop), β-lactamase gene (AmpR), which determines the resistance of E. coli cells transformed with plasmid pTEV-TMS to penicillin antibiotics, and XbaI / BamHI - DNA fragment containing the sequence of the TrxTEVrs-TMS hybrid gene, consisting of a fused in the frame 5′-terminal sequence encoding the N-terminal carrier protein from thioredoxin and a site for cleavage of TEV protease, and 3′-terminal sequence with optimized for expression in E. coli by codons encoding the C-terminal cleavable anti-angiogenic peptide TMS, and containing unique recognition sites of restriction endonucleases located at the following distance to the left of the BamHI site: SeaI - 583 bp, PvuI - 695 bp EcoRV - 3789 bp, XbaI - 5820 bp, BglII - 6308 bp, NcoI - 6340 bp. 2. Strain E. coli BL21 (DE3) / pTEV-TMS producing a TrxTEVrs-TMS hybrid polypeptide containing thioredoxin and an anti-angiogenic peptide, which is a modified fragment [L69K-95] of human tumstatin with Ser- sequences attached to the N-terminal and C-ends Gly-