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Prokaryotic Expression and Affinity Purification of DDX3 Protein.
Huang, Lan; Liang, Yue; Hou, Huijin; Tang, Min; Liu, Xinpeng; Ma, Yan-Ni; Liang, Shufang.
Afiliación
  • Huang L; 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.
  • Liang Y; 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.
  • Hou H; 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.
  • Tang M; 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.
  • Liu X; 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.
  • Ma YN; 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.
  • Liang S; 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.
Protein Pept Lett ; 31(3): 236-246, 2024.
Article en En | MEDLINE | ID: mdl-38303525
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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Cromatografía de Afinidad / Escherichia coli / ARN Helicasas DEAD-box Límite: Humans Idioma: En Revista: Protein Pept Lett Asunto de la revista: BIOQUIMICA Año: 2024 Tipo del documento: Article Pais de publicación: Países Bajos
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Cromatografía de Afinidad / Escherichia coli / ARN Helicasas DEAD-box Límite: Humans Idioma: En Revista: Protein Pept Lett Asunto de la revista: BIOQUIMICA Año: 2024 Tipo del documento: Article Pais de publicación: Países Bajos