Background: DDX3 is a protein with RNA helicase activity that is involved in a variety of biological processes, and it is an important protein target for the development of broad-spectrum antiviral drugs, multiple cancers and chronic inflammation. Objective: The objective of this study is to establish a simple and efficient method to express and purify DDX3 protein in E. coli, and the recombinant DDX3 should maintain helicase activity for further tailor-made screening and biochemical function validation. Methods: DDX3 cDNA was simultaneously cloned into pET28a-TEV and pNIC28-Bsa4 vectors and transfected into E. coli BL21 (DE3) to compare one suitable prokaryotic expression system. The 6×His-tag was fused to the C-terminus of DDX3 to form a His-tagging DDX3 fusion protein for subsequent purification. Protein dissolution buffer and purification washing conditions were optimized. The His-tagged DDX3 protein would bind with the Ni-NTA agarose by chelation and collected by affinity purification. The 6×His-tag fused with N-terminal DDX3 was eliminated from DDX3 by TEV digestion. A fine purification of DDX3 was performed by gel filtration chromatography. Results: The recombinant plasmid pNIC28-DDX3, which contained a 6×His-tag and one TEV cleavage site at the N terminal of DDX3 sequence, was constructed for DDX3 prokaryotic expression and affinity purification based on considering the good solubility of the recombinant His-tagging DDX3, especially under 0.5 mM IPTG incubation at 18 °C for 18 h to obtain more soluble DDX3 protein. Finally, the exogenous recombinant DDX3 protein was obtained with more than 95% purity by affinity purification on the Ni-NTA column and removal of miscellaneous through gel filtration chromatography. The finely-purified DDX3 still retained its ATPase activity. Conclusion: A prokaryotic expression pNIC28-DDX3 system is constructed for efficient expression and affinity purification of bioactive DDX3 protein in E. coli BL21(DE3), which provides an important high-throughput screening and validation of drugs targeting DDX3.

中文翻译：

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DDX3蛋白的原核表达及亲和纯化

背景：DDX3是一种具有RNA解旋酶活性的蛋白质，参与多种生物过程，是开发广谱抗病毒药物、多种癌症和慢性炎症的重要蛋白质靶点。目的：本研究的目的是建立一种简单、高效的大肠杆菌表达和纯化DDX3蛋白的方法，且重组DDX3应保持解旋酶活性，以便进一步进行定制筛选和生化功能验证。方法：将DDX3 cDNA同时克隆至pET28a-TEV和pNIC28-Bsa4载体中，并转染至大肠杆菌BL21（DE3）中，以比较一种合适的原核表达系统。将 6×His 标签融合到 DDX3 的 C 端，形成带有 His 标签的 DDX3 融合蛋白，用于后续纯化。优化了蛋白质溶解缓冲液和纯化洗涤条件。 His 标记的 DDX3 蛋白将通过螯合与 Ni-NTA 琼脂糖结合，并通过亲和纯化收集。通过 TEV 消化从 DDX3 中消除与 N 端 DDX3 融合的 6×His 标签。通过凝胶过滤色谱法对DDX3进行精细纯化。结果：考虑到重组His-tag的良好溶解性，构建了重组质粒pNIC28-DDX3，用于DDX3原核表达和亲和纯化，该质粒在DDX3序列的N端含有6×His-tag和1个TEV切割位点。标记DDX3，特别是在18℃下0.5 mM IPTG孵育18小时以获得更可溶的DDX3蛋白。最后通过Ni-NTA柱亲和纯化，凝胶过滤层析去除杂物，获得纯度95%以上的外源重组DDX3蛋白。精细纯化的 DDX3 仍保留其 ATP 酶活性。结论：构建了原核表达pNIC28-DDX3系统，用于在大肠杆菌BL21（DE3）中高效表达和亲和纯化具有生物活性的DDX3蛋白，为DDX3靶向药物的高通量筛选和验证提供了重要的依据。