Currently, purification step in the recombinant protein manufacture is still a great challenge and its cost far outweighs those of the upstream process. In this study, a functionalized cellulose-based monolith was constructed as an efficient affinity adsorbent for one-step purification of recombinant proteins. Firstly, the fundamental cellulose monolith (CE monolith) was fabricated based on thermally induced phase separation, followed by being modified with nitrilotriacetic acid anhydride through esterification to give NCE monolith. After chelating with Ni, the affinity adsorbent NCE-Ni monolith was obtained, which was demonstrated to possess a hierarchically porous morphology with a relatively high surface area, porosity and compressive strength. The adsorption behavior of NCE-Ni monolith towards β2-microglobulin with 6 N-terminus His-tag (His-β2M) was evaluated through batch and fixed-bed column experiments. The results revealed that NCE-Ni monolith exhibited a relatively fast His-β2M adsorption rate with a maximum adsorption capacity of 329.2 mg/g. The fixed-bed column adsorption implied that NCE-Ni monolith showed high efficiency for His-β2M adsorption. Finally, NCE-Ni monolith was demonstrated to have an excellent His-β2M purification ability from lysate with exceptional reusability. Therefore, the resultant NCE-Ni monolith had large potential to be used as an efficient adsorbent for recombinant protein purification in practical applications.

中文翻译：

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构建一种用于一步纯化重组蛋白的高效吸附剂：通过相分离方法制造的功能化纤维素基整体材料

目前，重组蛋白生产中的纯化步骤仍然是一个巨大的挑战，其成本远远超过上游过程。在这项研究中，构建了一种功能化的基于纤维素的整体材料作为一种有效的亲和吸附剂，用于重组蛋白的一步纯化。首先，基于热致相分离制备了基础纤维素整体料（CE整体料），然后用次氮基三乙酸酐通过酯化反应进行改性，得到NCE整体料。与Ni螯合后，得到了亲和吸附剂NCE-Ni整体结构，其具有分级多孔形态，具有较高的比表面积、孔隙率和抗压强度。通过批量和固定床柱实验评估了 NCE-Ni 整体材料对带有 6 个 N 末端 His 标签的 β2-微球蛋白 (His-β2M) 的吸附行为。结果表明，NCE-Ni整体柱表现出相对较快的His-β2M吸附速率，最大吸附容量为329.2 mg/g。固定床柱吸附表明 NCE-Ni 整体柱对 His-β2M 吸附表现出高效。最后，NCE-Ni 整体材料被证明具有出色的从裂解物中纯化 His-β2M 的能力，并且具有出色的可重复使用性。因此，所得的NCE-Ni整体材料在实际应用中具有作为重组蛋白纯化的有效吸附剂的巨大潜力。