| 中文简介: |
我们最近的研究挑战之一是利用蛋白质聚集的有效原理，涉及特定设计的蛋白质类似物的金属配位。专门设计的富含组氨酸的肿瘤坏死因子α（TNF-α）的类似物作为模型蛋白质。 LK801是一种TNF-α类似物，在钟形分子的尖端区域具有双组氨酸突变（Glu107HisGly108HisTNF-α）。由于对称的三聚体结构，尖端区域中的组氨酸残基形成几乎平面的六个易接近的组氨酸簇，导致与固定化金属亲和色谱（IMAC）的强结合，其用于有效的单步纯化。 IMAC还用于制备His10-TNF，一种具有His10标签的TNF-α类似物和负责肠激酶切割的氨基酸序列，其添加到天然N末端和H7dN6TNF类似物，其在N-末端具有由七个组氨酸组成的标签。 。制备两种含金属离子的无机纳米粒子，通过沉淀法制备磷酸锌纳米粒子，并通过在市售二氧化硅纳米粒子上吸附锌离子制备锌改性二氧化硅。为了概念证明，在初步实验中使用牛血清白蛋白（BSA）进行结合。 BSA含有天然表面暴露的组氨酸，并且还预期与金属离子协同结合纳米颗粒。当这些颗粒暴露于低pH缓冲液或含有咪唑的缓冲液时，通过SDS-PAGE分析证实了BSA的释放，从而证明了金属特异性结合的可逆性。在接下来的步骤中，富含组氨酸的TNF类似物用于结合Zn-磷酸盐纳米颗粒，并且在不同条件下进行释放研究。我们还在结合之前和之后从无机纳米颗粒中释放后测量TNF-α类似物的生物活性。通过使用金属离子（Zn2 +）的蛋白质自组装来尝试控制TNF-α类似物纳米颗粒的形成。仅添加锌离子进行第一次实验，随后我们使用Zn2 +和不同的生物相容性螯合物（植酸和1,4,8,11-四氮杂环十四烷-1,4,8,11-四乙酸）进行实验。在向测试动物施用上述蛋白质聚集体后，预期增加的免疫应答。在TNF-α类似物的情况下，增强抗TNF-α抗体的形成将有利于作为开发用于与病原性升高的TNF-α水平相关的慢性疾病的新药的基础（类风湿性关节炎，克罗恩病，牛皮癣等）。 ）。 His10-TNF类似物显得特别有趣，因为它表现出非常低的体外细胞毒活性。在形成纳米结构后，预期可接近的受体结合位点的数量显着减少并因此甚至更低的细胞毒性，导致抗TNF-α抗体的安全形成。
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| 课程简介: |
One of our recent research challenges has been exploitation of the active principle of protein aggregation, involving metal coordination of specifically designed protein analogues. Specifically designed analogues of tumour necrosis factor alpha (TNF-alpha) rich in histidines served as model proteins. LK801 is a TNF-alpha analogue with double histidine mutation (Glu107HisGly108HisTNF-alpha) in the tip region of the bell-shaped molecule. Due to the symmetrical trimeric structure, histidine residues in the tip region form an almost planar cluster of six well accessible histidines resulting in strong binding to Immobilized Metal Affinity Chromatography (IMAC), which was used for efficient single step purification. IMAC was also used for preparation of His10-TNF, a TNF-alpha analogue with His10 tag and amino acid sequence responsible for enterokinase cleavage added to the native N-terminus and H7dN6TNF analogue, which has a tag comprised of seven histidines on N-terminus. Two types of inorganic nanoparticles containing metal ions were prepared, zinc phosphate nanoparticles by precipitation, and zinc modified silica by adsorption of zinc ions on commercially available silica nanoparticles. For the proof of concept, in preliminary experiments bovine serum albumin (BSA) was used for binding. BSA contains naturally surface exposed histidines and was also expected to coordinatively bind to nanoparticles with metal ions. When such particles were exposed to low pH buffers or buffers containing imidazole, release of BSA was confirmed by SDS-PAGE analysis, thus proving the reversibility of metal-specific binding. In the next steps, histidine rich TNF analogues were used for binding to Zn-phosphate nanoparticles and release studies were performed under different conditions. We also measured biological activity of TNF-alpha analogues prior to binding and later after the release from inorganic nanoparticles. The controlled formation of TNF-alpha analogues nanoparticles was tried by protein self-assembly using metal ions (Zn2+). First experiments were performed with only the addition of zinc ions and later we performed experiments using Zn2+ and different biocompatible chelates (phytic acid and 1,4,8,11-Tetraazacyclotetradecane-1,4,8,11-tetraacetic acid). Upon administration of above mentioned protein aggregates to the testing animals, an increased immune response is anticipated. In the case of TNF-alpha analogues, an enhanced formation of antibodies against TNF-alpha would be advantageous serving as a basis for developing new drugs for chronic diseases associated with pathogenically elevated TNF-alpha levels (rheumatoid arthritis, Crohn disease, psoriasis, etc.). His10-TNF analogue appears especially interesting, since it exhibits very low in vitro cytotoxic activity. Upon formation of nanostructures, a significantly diminished number of accessible receptor binding sites and consequently even more reduced cytotoxicity is expected, leading to safe formation of anti-TNF-alpha antibodies.
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