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

Citation:

PDF (1795 KB)

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)

More Information

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

Graphical Abstract

Abstract

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

FullText(HTML)

References (15)

References

[1]	. Chem Soc Rev， 2018， 47（24）： 9046-9068.
[2]	Shah NH， Muir TW. Inteins： nature''''s gift to protein chemists［J］. Chem Sci， 2014， 5（1）： 446-461.
[3]	Shi SW， Chen HH， Jiang H， et al. A novel self-cleavable tag Zbasic-?I-CM and its application in the soluble expression of recombinant human interleukin-15 in Escherichia coli［J］. Appl Microbiol Biotechnol， 2017， 101（3）： 1133-1142.
[4]	Zettler J， Schütz V， Mootz HD. The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction［J］. FEBS Lett， 2009， 583（5）： 909-914.
[5]	Iwai H， Züger S， Jin J， et al. Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme［J］. FEBS Lett， 2006， 580（7）： 1853-1858.
[6]	Ramirez M， Valdes N， Guan DL， et al. Engineering split intein DnaE from Nostoc punctiforme for rapid protein purification［J］. Protein Eng Des Sel， 2013， 26（3）： 215-223.
[7]	Guan DL， Ramirez M， Chen ZL. Split intein mediated ultra-rapid purification of tagless protein （SIRP）［J］. Biotechnol Bioeng， 2013， 110（9）： 2471-2481.
[8]	Shah NH， Eryilmaz E， Cowburn D， et al. Naturally split inteins assemble through a “capture and collapse” mechanism［J］. J Am Chem Soc， 2013， 135（49）： 18673-18681.
[9]	Stevens AJ， Sekar G， Gramespacher JA， et al. An atypical mechanism of split intein molecular recognition and folding［J］. J Am Chem Soc， 2018， 140（37）： 11791-11799.
[10]	Shah NH， Vila-Perelló M， Muir TW. Kinetic control of one-pot trans-splicing reactions by using a wild-type and designed split intein［J］. Angew Chem Int Ed Engl， 2011， 50（29）： 6511-6515.
[11]	Wang J， Han L， Chen JS， et al. Reduction of non-specific toxicity of immunotoxin by intein mediated reconstitution on target cells［J］. Int Immunopharmacol， 2019， 66： 288-295.
[12]	Pirzer T， Becher KS， Rieker M， et al. Generation of potent anti-HER1/2 immunotoxins by protein ligation using split inteins［J］. ACS Chem Biol， 2018， 13（8）： 2058-2066.
[13]	Han L， Zong HF， Zhou YX， et al. Naturally split intein Npu DnaE mediated rapid generation of bispecific IgG antibodies［J］. Methods， 2019， 154： 32-37.
[14]	Vila-Perelló M， Liu ZH， Shah NH， et al. StreamLined expressed protein ligation using split inteins［J］. J Am Chem Soc， 2013， 135（1）： 286-292.
[15]	Lu W， Sun ZY， Tang YC， et al. Split intein facilitated tag affinity purification for recombinant proteins with controllable tag removal by inducible auto-cleavage［J］. J Chromatogr A， 2011， 1218（18）： 2553-2560.

Cited By

Cited by

Periodical cited type(4)

1.	肖咪，罗蓉，赵红玉，苏彦奇，李丹，蔡旭，余涛. 超声波辅助低共熔溶剂提取淫羊藿总黄酮的工艺优化及其降糖活性研究. 中国医院药学杂志. 2025(02): 135-140 .
2.	王丹，林鹏，刘蓉，刘芳，张鹏，袁昕蓉. 复方甘乐注射液对小鼠血糖的影响研究. 品牌与标准化. 2024(02): 21-23+27 .
3.	申凤霞，唐云峰，赵利枝，严琴琴，周冰，马云，李倩，关永霞，范建伟. 心通制剂“异病同治”冠心病和糖尿病的作用机制研究. 巴楚医学. 2024(04): 54-61 .
4.	董国华，杜银苹，耿猛，李菲，董国梁. antagomiR-21上调SIRT1激活PI3K/Akt/eNOS信号通路改善T2DM大鼠冠状动脉内皮依赖性舒张. 中国动脉硬化杂志. 2023(09): 771-778 .

Other cited types(1)

Get Citation

PDF

XML

Article views (150) PDF downloads (679) Cited by(5)

Turn off MathJax

Article Contents

Abstract

References

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

Abstract

References

Cited by

Periodical cited type(4)

Other cited types(1)

Catalog

Export File

Citation

Format

Content