College of Medical Laboratory, Dalian Medical University, Dalian, China.
Institute of Cancer Stem Cell, Dalian Medical University, Dalian, China.
J Sep Sci. 2024 Sep;47(18):e202400471. doi: 10.1002/jssc.202400471.
Recombinant proteins hold significant importance in numerous disciplines. As the demand for expressing and purifying these proteins grows, the scientific community is in dire need of a simple yet versatile methodology that can efficiently purify these proteins. Aptamers as synthetic nucleic acid-based ligands with high affinity have shown promise in this regard, as they can capture targets through molecular recognition. In this study, novel aptamer-functionalized polydopamine-coated magnetic graphene oxide nanocomposites were facilely prepared, achieving an impressive average aptamer coverage density (45 nmol/mg). These nanocomposites exhibited a uniform structure and robust magnetic responsiveness. The findings indicated that they possess several advantages, such as rapid adsorption, substantial capacity (171.4 mg/g), and excellent reusability. Notably, due to the inherent properties of nucleic acids, the immobilized aptamer-magnetic beads can be utilized repeatedly with high purification efficiency. Finally, the nanocomposites were further employed to purify His-tagged proteins from actual samples. Remarkably, they were able to selectively and efficiently isolate His-tagged retinoid X receptor alpha protein from complex Escherichia coli lysate. The purified His-tagged retinoid X receptor alpha protein was analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This confirmed the efficacy of developed nanocomposites, reinforcing their vast potential for purification of His-tagged recombinant proteins.
重组蛋白在许多领域都具有重要意义。随着表达和纯化这些蛋白质的需求不断增长，科学界迫切需要一种简单而通用的方法，能够有效地纯化这些蛋白质。适体作为具有高亲和力的合成核酸配体，在这方面显示出了潜力，因为它们可以通过分子识别捕获靶标。在这项研究中，我们简便地制备了新型适体功能化的聚多巴胺包覆的磁性氧化石墨烯纳米复合材料，实现了令人印象深刻的平均适体覆盖率（45 nmol/mg）。这些纳米复合材料具有均匀的结构和强大的磁响应性。研究结果表明，它们具有快速吸附、大容量（171.4 mg/g）和出色的可重复使用性等优点。值得注意的是，由于核酸的固有特性，固定化的适体-磁性珠可以反复使用，并且具有很高的纯化效率。最后，我们进一步将纳米复合材料用于从实际样品中纯化 His 标记的蛋白质。令人瞩目的是，它们能够从复杂的大肠杆菌裂解物中选择性和有效地分离 His 标记的视黄醇 X 受体 alpha 蛋白。使用基质辅助激光解吸/电离飞行时间质谱分析了纯化的 His 标记的视黄醇 X 受体 alpha 蛋白。这证实了所开发的纳米复合材料的功效，增强了它们在纯化 His 标记的重组蛋白方面的巨大潜力。
