A thermo-responsive, self-assembling biointerface for on demand release of surface-immobilised proteins.
A thermo-responsive, self-assembling biointerface for on demand release of surface-immobilised proteins.
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DOI：
10.1039/c9bm01957j
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发表时间：
2020-04
影响因子：
6.6
通讯作者：
Angela Saccardo;Mikhail Soloviev;Enrico Ferrari
中科院分区：
文献类型：
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作者：
Angela Saccardo;Mikhail Soloviev;Enrico Ferrari
药物输送应用、分析设备中使用的可切换表面的合成以及具有复杂结构和功能的下一代生物材料的组装需要专用化学物质在固液界面按需捕获和释放生物分子。在这里，我们报告了二元自组装多肽系统的工程，用于可逆蛋白质捕获、固定和从固体表面受控的热响应释放。二元系统的第一个元素是固定在固体表面上的通用蛋白质底物。该蛋白质的生物启发来自神经元 SNAP25，SNAP25 是参与突触小泡与突触膜对接和融合的蛋白质。第二个元件是人工嵌合蛋白，经过工程改造，包含来自三种不同蛋白的不同结构域：突触蛋白、VAMP 和 SNAP25。这些天然蛋白质构成了真核生物中专门用于囊泡运输的机制。我们从这些蛋白质中去除了大约 70% 的天然蛋白质序列，并构建了能够与固定基质进行高亲和力相互作用和自组装的蛋白质嵌合体。工程蛋白质复合物的两部分之间的相互作用很强，但完全可逆，因此嵌合体可以作为标签重组融合到感兴趣的蛋白质上，以允许在预定温度下加热时自发组装和刺激敏感地从表面释放。据报道，有两种热响应标签：第一种具有显着的热稳定性，熔化温度约为 80°C；第二种具有出色的热稳定性，其熔化温度约为 80°C。第二个在大约 45°C 的较低温度下分解。后者是治疗性蛋白质远程控制局部递送的有前途的候选者，因为使用磁场、红外光或聚焦超声波可以实现生理上可耐受的局部温度升高至 40-45°C 范围。重要的是，这两种新型多肽为未来功能性蛋白质的工程设计提供了更广泛的蓝图，这些蛋白质具有可预测的折叠和对外部刺激的反应。
Dedicated chemistries for on-demand capture and release of biomolecules at the solid-liquid interface are required for applications in drug delivery, for the synthesis of switchable surfaces used in analytical devices and for the assembly of next-generation biomaterials with complex architectures and functions. Here we report the engineering of a binary self-assembling polypeptide system for reversible protein capture, immobilisation and controlled thermo-responsive release from a solid surface. The first element of the binary system is a universal protein substrate immobilised on a solid surface. This protein is bio-inspired by the neuronal SNAP25, which is the protein involved in the docking and fusion of synaptic vesicles to the synaptic membrane. The second element is an artificial chimeric protein engineered to include distinct domains from three different proteins: Syntaxin, VAMP and SNAP25. These native proteins constitute the machinery dedicated to vesicle trafficking in eukaryotes. We removed approximately 70% of native protein sequence from these proteins and constructed a protein chimera capable of high affinity interaction and self-assembly with immobilised substrate. The interaction of the two parts of the engineered protein complex is strong but fully-reversible and therefore the chimera can be recombinantly fused as a tag to a protein of interest, to allow spontaneous assembly and stimuli-sensitive release from the surface upon heating at a predetermined temperature. Two thermo-responsive tags are reported: the first presents remarkable thermal stability with melting temperature of the order of 80 °C; the second disassembles at a substantially lower temperature of about 45 °C. The latter is a promising candidate for remote-controlled localised delivery of therapeutic proteins, as physiologically tolerable local increase of temperatures in the 40-45 °C range can be achieved using magnetic fields, infra-red light or focused ultrasound. Importantly, these two novel polypeptides provide a broader blueprint for the engineering of future functional proteins with predictable folding and response to external stimuli.