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Optimized Expression of Recombinant Human NIMA-related Kinase 7 (NEK7) with A Higher Purity in Escherichia coli

Author(s):

Xing-Jie Zhang, Ting-Ting Wang, Yu-Kun Pu, Lin Zeng, Rui-Han Zhang, Xiao-Li Li, Xu Ji* and Wei-Lie Xiao*   Pages 1 - 7 ( 7 )

Abstract:


Background: NIMA (never in mitosis, gene A) serine/threonine kinase 7 (NEK7) is a regulator of mitosis spindle in mammals and is considered as a drug target of inflammasome related inflammatory diseases. However, most commercially available or reported recombinant NEK7 proteins are either inactive or have low purity. These shortcomings limit the pharmacological studies and development of NEK7 inhibitors.

Objective: To elucidate what causes the NEK7 low purity in E. coli, and optimize a protocol to improve the protein purity.

Methods: A comparative study of expression full length NEK7 with an N-terminal His-tag or a Cterminal His-tag was performed. His-affinity resin, ion exchange and gel filtration chromatography were used to purify NEK7. The protein was identified by mass spectrometry. The activity and folding of NEK7 were evaluated by chemiluminescent assay and thermal shift assay.

Results: Our results demonstrated that N-terminal tagged protein was toxic to E. coli, resulting in incomplete translated products. The C-terminal tagged NEK7-His6 had a much higher purity than that of an N-terminal tag. The Ni2+ resin one-step purification led to a purity of 91.7%, meeting the criteria of most kinase assays. With two-step and three-step procedures, the protein purities were 94.7% and ~100%, respectively. The NEK7 purified in this work maintained its kinase activity and correct conformation, and the compound-protein interaction ability.

Conclusion: Our optimized protocol could produce good purity of His tagged NEK7 in E. coli, and the kinase activity and biophysical characteristics of which are preserved.

Keywords:

NEK7, histidine tag, expression, purification, high purity, kinase.

Affiliation:

Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education; Yunnan Provincial Center for Research & Development of Natural Products; School of Chemical Science and Technology, Yunnan University, Kunming, 650091, Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education; Yunnan Provincial Center for Research & Development of Natural Products; School of Chemical Science and Technology, Yunnan University, Kunming, 650091, Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education; Yunnan Provincial Center for Research & Development of Natural Products; School of Chemical Science and Technology, Yunnan University, Kunming, 650091, Public Technology Service Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, 650032, Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education; Yunnan Provincial Center for Research & Development of Natural Products; School of Chemical Science and Technology, Yunnan University, Kunming, 650091, Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education; Yunnan Provincial Center for Research & Development of Natural Products; School of Chemical Science and Technology, Yunnan University, Kunming, 650091, Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education; Yunnan Provincial Center for Research & Development of Natural Products; School of Chemical Science and Technology, Yunnan University, Kunming, 650091, Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education; Yunnan Provincial Center for Research & Development of Natural Products; School of Chemical Science and Technology, Yunnan University, Kunming, 650091



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