重组DNA
大肠杆菌
化学
激酶
蛋白激酶A
赫拉
生物化学
分子生物学
细胞生物学
生物
细胞
基因
作者
Xing‐Jie Zhang,Tingting Wang,Yu‐Kun Pu,Lin Zeng,Ruihan Zhang,Xiao‐Li Li,Xu Ji,Wei‐Lie Xiao
出处
期刊:Protein and Peptide Letters
[Bentham Science]
日期:2021-12-01
卷期号:28 (12): 1391-1397
标识
DOI:10.2174/0929866528666211118092410
摘要
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.To elucidate what causes the NEK7 low purity in E. coli, and optimize a protocol to improve the protein purity.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.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.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.
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