School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, 950 Atlantic Drive NW, Atlanta, Georgia, 30332, USA.
BioEngineering Program, Georgia Institute of Technology, 950 Atlantic Drive NW, Atlanta, Georgia, 30332, USA.
J Mater Chem B. 2023 Jul 12;11(27):6443-6452. doi: 10.1039/d3tb00200d.
Protein vesicles made from bioactive proteins have potential value in drug delivery, biocatalysis, and as artificial cells. As the proteins are produced recombinantly, the ability to precisely tune the protein sequence provides control not possible with polymeric vesicles. The tunability and biocompatibility motivated this work to develop protein vesicles using rationally designed protein building blocks to investigate how protein sequence influences vesicle self-assembly and properties. We have reported an elastin-like polypeptide (ELP) fused to an arginine-rich leucine zipper (Z) and functional, globular proteins fused to a glutamate-rich leucine zipper (Z) that self-assemble into protein vesicles when warmed from 4 to 25 °C due to the hydrophobic transition of ELP. Previously, we demonstrated the ability to tune vesicle properties by changing protein and salt concentration, Z : Z ratio, and warming rate. However, there is a limit to the properties that can be achieved assembly conditions. In order to access a wider range of vesicle diameter and stability profiles, this work investigated how modifiying the hydrophobicity and length of the ELP sequence influenced self-assembly and the final properties of protein vesicles using mCherry as a model globular protein. The results showed that both transition temperature and diameter of protein vesicles were inversely correlated to the ELP guest residue hydrophobicity and the number of ELP pentapeptide repeats. Additionally, sequence manipulation enabled assembly of vesicles with properties not accessible by changes to assembly conditions. For example, introduction of tyrosine at 5 guest residue positions in ELP enabled formation of nanoscale vesicles stable at physiological salt concentration. This work yields design guidelines for modifying the ELP sequence to manipulate protein vesicle transition temperature, size and stability to achieve desired properties for particular biofunctional applications.
由生物活性蛋白制成的蛋白囊泡在药物传递、生物催化和人工细胞方面具有潜在价值。由于蛋白质是通过重组生产的，因此能够精确调整蛋白质序列提供了使用聚合囊泡无法实现的控制。这种可调节性和生物相容性促使我们使用合理设计的蛋白质构建块来开发蛋白囊泡，以研究蛋白质序列如何影响囊泡自组装和性质。我们已经报道了一种弹性蛋白样多肽（ELP）与富含精氨酸的亮氨酸拉链（Z）融合，以及功能球状蛋白与富含谷氨酸的亮氨酸拉链（Z）融合，由于 ELP 的疏水性转变，这些蛋白在从 4 到 25°C 加热时自组装成蛋白囊泡。此前，我们证明了通过改变蛋白质和盐浓度、Z：Z 比和升温速率来调节囊泡性质的能力。然而，在组装条件下，能够实现的性质是有限的。为了获得更广泛的囊泡直径和稳定性分布，这项工作研究了改变 ELP 序列的疏水性和长度如何影响自组装以及使用 mCherry 作为模型球状蛋白的蛋白囊泡的最终性质。结果表明，蛋白囊泡的相变温度和直径与 ELP 客体残基疏水性和 ELP 五肽重复数成反比。此外，序列操作使具有通过改变组装条件无法获得的性质的囊泡组装成为可能。例如，在 ELP 的 5 个客体残基位置引入酪氨酸可形成在生理盐浓度下稳定的纳米级囊泡。这项工作为修饰 ELP 序列以操纵蛋白囊泡相变温度、大小和稳定性提供了设计准则，以实现特定生物功能应用所需的性质。
