Improving proteolytic stability of <i>Npu</i> DnaE C-fragment in <i>Escherichia coli</i> expression system

CHEN Hao; CAO Jin; ZHANG Jing; ZHU Jianwei; CHEN Junsheng

doi:10.11665/j.issn.1000-5048.20200312

Journal of China Pharmaceutical University > 2020 > 51(3): 340-348. > DOI: 10.11665/j.issn.1000-5048.20200312

CHEN Hao, CAO Jin, ZHANG Jing, ZHU Jianwei, CHEN Junsheng. Improving proteolytic stability of Npu DnaE C-fragment in Escherichia coli expression system[J]. Journal of China Pharmaceutical University, 2020, 51(3): 340-348. DOI: 10.11665/j.issn.1000-5048.20200312

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Improving proteolytic stability of Npu DnaE C-fragment in Escherichia coli expression system

Funds: This study was supported by the National Natural Science Foundation of China (No.81773621)

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Received Date: February 21, 2020
Revised Date: May 12, 2020

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Abstract

Naturally split Npu DnaE intein can mediate rapid trans-splicing and C-cleavage, which is of great use in many aspects of protein engineering. However, the degradation of NpuC during expression and purification reduces the yield and purity of recombinant protein. N2C, an extended NpuN2-containing N-terminal NpuC fragment, was constructed to improve NpuC stability. N2C was expressed in BL21(DE3) and purified by affinity chromatography. The degradation ratio was calculated by ImageJ, and the factors affecting the C-terminal cleavage reaction of intein, such as temperature, DTT concentration and N/C ratio, were also investigated. The results showed that N2C lowered the proportion of degradation to 2.7%-7.2% and the yield of C-terminal cleavage reached 90% in 30 min at 37 °C with an N/C ratio of 5∶1 catalyzed by 1 mmol/L DTT. N2C can not only improve the stability of NpuC in Escherichia coli expression system, but also retain the activity of C-terminal cleavage reaction, which is of great significance for its application in protein purification.
- Npu DnaE,
- C-terminal cleavage,
- protein stability,
- protein degradation

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