Prokaryotic Expression and Affinity Purification of DDX3 Protein

Title:Prokaryotic Expression and Affinity Purification of DDX3 Protein

Volume: 31 Issue: 3

Author(s): Lan Huang, Yue Liang, Huijin Hou, Min Tang, Xinpeng Liu, Yan-ni Ma and Shufang Liang*

Affiliation:

• Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, No.17, Section 3 of Renmin South Road, Chengdu, Sichuan, 610041, P.R. China

Keywords: DDX3, Prokaryotic expression, affinity purification, ATPase activity, His-tag, Ni-NTA column.

Abstract:

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.

Objectives: 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.

Cite this article as:

Huang Lan, Liang Yue, Hou Huijin, Tang Min, Liu Xinpeng, Ma Yan-ni and Liang Shufang*, Prokaryotic Expression and Affinity Purification of DDX3 Protein, Protein & Peptide Letters 2024; 31 (3) . https://dx.doi.org/10.2174/0109298665285625231222075700