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Prokaryotic Expression and Affinity Purification of DDX3 Protein
Lan Huang1, Yue Liang1, Huijin Hou1
1Department 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.
Protein and Peptide Letters
|February 2, 2024
Summary
Researchers developed an efficient method to express and purify DDX3 protein in E. coli. The resulting recombinant DDX3 protein maintains its helicase activity, crucial for drug development targeting viral infections and cancers.
Area of Science:
- Molecular Biology
- Biochemistry
Background:
- DDX3 is a protein with RNA helicase activity implicated in various biological processes.
- It serves as a key target for developing broad-spectrum antiviral drugs, anti-cancer therapies, and anti-inflammatory treatments.
Purpose of the Study:
- To establish a simple and efficient method for expressing and purifying DDX3 protein in E. coli.
- To ensure the recombinant DDX3 protein retains its helicase activity for subsequent drug screening and functional validation.
Main Methods:
- DDX3 cDNA was cloned into pET28a-TEV and pNIC28-Bsa4 vectors for expression in E. coli BL21 (DE3).
- A 6×His-tag was used for affinity purification, followed by TEV digestion to remove the tag.
- Optimization of protein dissolution and purification conditions, including affinity chromatography and gel filtration.
Main Results:
- The pNIC28-DDX3 plasmid facilitated efficient prokaryotic expression and purification.
- Optimal conditions (0.5 mM IPTG, 18°C for 18 h) yielded highly soluble recombinant His-tagged DDX3.
- Purified DDX3 achieved >95% purity and retained ATPase activity.
Conclusions:
- A robust prokaryotic expression system (pNIC28-DDX3 in E. coli BL21 (DE3)) was successfully established.
- This system enables efficient expression and purification of bioactive DDX3 protein.
- The developed method supports high-throughput screening and validation of drugs targeting DDX3.
