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.
