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.
计量
- 文章访问数:
- HTML全文浏览量: 0
- PDF下载量:
下载:
