断裂内含肽C片段在大肠埃希菌表达系统中酶解稳定性的改良
Improving proteolytic stability of Npu DnaE C-fragment in Escherichia coli expression system
-
摘要: 断裂内含肽Npu DnaE介导的蛋白质剪接、剪切反应,可以应用于蛋白质工程领域诸多方面,但其C段重组蛋白在表达纯化过程中发生的降解,降低了重组蛋白的产率和纯度。为提高NpuC段稳定性,本研究构建了N端融合NpuN2片段的NpuC延长变体N2C。将N2C在BL21(DE3)中进行表达、用亲和色谱进行纯化,用ImageJ扫描计算表达纯化中降解情况,进而对影响内含肽C端剪切反应的各因素如温度、DTT浓度、N/C比例等进行了考察。结果表明,延长变体N2C使降解产物占比降低至2.7%~7.2%,在1 mmol/L DTT催化,N/C比例为5∶1,37 ℃反应条件下,30 min产物生成率达90%。N2C在提升Npu DnaE内含肽C段在大肠埃希菌表达系统中表达、纯化过程的稳定性的同时,保留了其C端剪切反应的活性,对其在蛋白纯化领域应用有重要意义。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.
-
Keywords:
- Npu DnaE /
- C-terminal cleavage /
- protein stability /
- protein degradation
-
-
[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. -
期刊类型引用(16)
1. 庄件兵,朱莉,周璐,王明明. UPLC-MS/MS法快速测定污水中多种化学毒品残留. 化学工程师. 2025(04): 28-33+38 . 百度学术
2. 李昕怡,王韬任,牛德云,徐玉,李斌,孙加学,薛丹,李虹. UPLC-MS/MS法检测污水中4种合成大麻素及其代谢产物. 中国法医学杂志. 2025(02): 213-219 . 百度学术
3. 郑吴淇,宁弘宇,陈昊,黄忠平,范一雷,柯星. 流动注射-串联质谱法分析污水中11种毒品. 分析试验室. 2024(05): 705-710 . 百度学术
4. 刘昕,王兵益,杨发震. 水环境毒品监测用于毒情评估的标准体系研究. 云南警官学院学报. 2023(04): 7-12 . 百度学术
5. 彭诗琪,赵嘉辉,赖华杰,桑柳波. 基于阳离子交换的固相萃取与液相色谱—串联质谱法联用分析污水中的17种非法药物. 化学研究与应用. 2023(08): 1956-1965 . 百度学术
6. 李雪蕾,袁健彪. 浅谈生活污水中毒品检测技术的分析和应用. 中国石油和化工标准与质量. 2022(04): 41-43 . 百度学术
7. 郭晶晶,陈丹萍,董露斌,杨飞,胡双英. SPE-HPLC-ESI-MS/MS检测污水中常见13种违禁药物的方法. 新型工业化. 2022(04): 51-54+58 . 百度学术
8. 王叶,徐磊,徐鹏,杭太俊,宋敏,王优美,徐慧. 污水中常见毒品的分析方法优化及验证. 中国药科大学学报. 2022(04): 467-472 . 本站查看
9. 李雪松. 生活污水中滥用药物检测技术的应用与分析. 生物化工. 2022(04): 58-61 . 百度学术
10. 王欢博,米兰,霍婷婷,唐恬,徐布一. 大气环境中毒品监测研究进展. 环境化学. 2022(09): 2974-2985 . 百度学术
11. 王平,刘晓云,郑振成,梁桂巧,赖胜强. 应用固相萃取-超高效液相色谱-串联质谱法同时检测城市污水中氟胺酮及2种位置异构体. 中国司法鉴定. 2022(05): 67-72 . 百度学术
12. 向平. 污水毒品监测技术:进展、挑战与展望. 中国司法鉴定. 2022(05): 17-21 . 百度学术
13. 丁艳,乔宏伟,陈捷,张婷婷,花镇东,杭太俊,刘培培. 在线固相萃取-超高效液相色谱-串联质谱法同时检测污水中氟胺酮等21种毒品及其代谢物. 中国司法鉴定. 2022(05): 39-50 . 百度学术
14. 赵明明,刘冬娴,伍岚,刘炜,贺江南,陈志伟,易荣楠. 固相萃取/液质联用法检测污水中14种毒品及代谢物. 中国给水排水. 2022(24): 133-138 . 百度学术
15. 王美丽,李敦毅. QuEChERS法提取-液相色谱-质谱法检测分析制药园区污水中青霉素、洁霉素、土霉素、四环素和庆大霉素残留方法的建立. 分析仪器. 2021(04): 150-154 . 百度学术
16. Jingyuan Wang,Likai Qia,Chenzhi Hou,Tingting Zhang,Mengyi Chen,Haitao Meng,Mengxiang Su,Hui Xu,Zhendong Hua,Youmei Wang,Bin Di. Automatic analytical approach for the determination of 12 illicit drugs and nicotine metabolites in wastewater using on-line SPE-UHPLC-MS/MS. Journal of Pharmaceutical Analysis. 2021(06): 739-745 . 必应学术
其他类型引用(2)
计量
- 文章访问数: 167
- HTML全文浏览量: 4
- PDF下载量: 679
- 被引次数: 18