BIFUNCTIONAL PROTEIN FOR BINDING TELOMERIC DNA AND MAMMALIAN CELLS

用于结合端粒 DNA 和哺乳动物细胞的双功能蛋白

• 批准号: 6162598
• 负责人: M ROSENFELD
• 金额: --
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6162598
• 关键词: DNA binding protein; binding proteins; chimeric proteins; epidermal growth factor; growth factor receptors; nucleic acid repetitive sequence; telomere; tissue /cell culture; transfection; transfection /expression vector

We have developed a system to deliver DNA to mammalian cells utilizing a protein with specific receptor and DNA binding properties. The fusion protein was constructed with transforming growth factor a (TGFa) at the N-terminus and human telomeric repeat binding factor (hTRF) at the C-terminus (TGFa/TRF), expressed in E.coli, refolded from inclusion bodies and purified through ion exchange columns and a telomeric DNA (T2AG3 repeats)-specific affinity column. Binding to telomeric DNA was evident by mobility shift assay. TGFa binding activity to the epidermal growth factor (EGF) receptor was confirmed by competition studies whereby TGFa/TRF displaced 125I-EGF from EGF receptors. Plasmid DNA containing T2AG3 repeats and the E. coli Lac Z (beta-gal) reporter gene was bound to TGFa/TRF and the complexes were condensed by poly-L-lysine (pL), and added to cells. beta-gal expression was observed in EGF expressing cells above the background of pL alone and this increase inbeta-gal expression was blocked in the presence of excess EGF protein. Current work focuses on confirming specificity and improving efficiency by utilizing modified fusion proteins incorporating translocation domains to enhance intracellular trafficking of DNA to the nucleus. We will test this approach in vivo in mice and assess the feasibility of TGFa/TRF to deliver human artificial chromosomes into human cells.

我们已经开发了一个用于利用哺乳动物细胞的DNA的系统 具有特定受体和DNA结合特性的蛋白质。融合 蛋白质是用转化生长因子A（TGFA）在 N末端和人端粒重复结合因子（HTRF） C-terminus（TGFA/TRF），在e.coli中表达，从包含中重折叠 身体并通过离子交换柱和端粒DNA纯化 （T2AG3重复） - 特定亲和力列。与端粒DNA结合 由移动性转移测定法明显。 TGFA结合活性与表皮 竞争研究证实了生长因子（EGF）受体 TGFA/TRF从EGF受体中移动125-EGF。含质粒DNA T2AG3重复和大肠杆菌lac Z（beta-gal）报告基因结合了 通过聚-l-赖氨酸（PL）凝结TGFA/TRF和复合物 添加到细胞中。在EGF表达细胞中观察到β-gal表达 在单独使用PL的背景之上，并增加Inbeta-gal表达 在存在过量的EGF蛋白的情况下被阻塞。目前的工作重点 通过利用修改后确认特异性并提高效率 融合易位域以增强的融合蛋白 DNA向细胞核的细胞内运输。我们将测试这个 在小鼠中的体内接近，并评估TGFA/TRF的可行性 将人类人造染色体输送到人类细胞中。