Intein-mediated Expression in <i>E. coli</i> and Amidation of Antimicrobial peptide MME

YE Ruobai; WU Zhenhong; MIAO Xiaoqing

doi:10.19303/j.issn.1008-0384.2020.11.013

Volume 35 Issue 11

Nov. 2020

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Article Navigation > Fujian Journal of Agricultural Sciences > 2020 > 35(11): 1265-1270

YE R B, WU Z H, MIAO X Q. Intein-mediated Expression in E. coli and Amidation of Antimicrobial peptide MME [J]. Fujian Journal of Agricultural Sciences，2020，35（11）：1265−1270 doi: 10.19303/j.issn.1008-0384.2020.11.013

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YE R B, WU Z H, MIAO X Q. Intein-mediated Expression in E. coli and Amidation of Antimicrobial peptide MME [J]. Fujian Journal of Agricultural Sciences，2020，35（11）：1265−1270 doi: 10.19303/j.issn.1008-0384.2020.11.013

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Intein-mediated Expression in E. coli and Amidation of Antimicrobial peptide MME

doi: 10.19303/j.issn.1008-0384.2020.11.013

YE Ruobai^1
,,
WU Zhenhong²,
MIAO Xiaoqing^{1, 2
,
,}

1.
College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China
2.
College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China

Received Date: 2020-10-10
Rev Recd Date: 2020-11-05

Available Online: 2021-01-26

Publish Date: 2020-11-30

Abstract

Abstract

Objective Efficient method to amidate the carbon-terminal of the antimicrobial peptide MME through self-splitting intein in recombination fusion proteins expressed in E. coli with DTT in the presences of MESNa and NH₄HCO₃ was explored. Method Recombinant plasmid of MME was constructed to induce the intein-mediated expression in E. coli to successively obtain fusion proteins composed of histidine, sumo label, target polypeptide, and intein. The proteins were subsequently purified in a process using a nickel column and dialysis. With MESNa and NH₄HCO₃, an intein self-split procedure was completed by DTT to amidate the carbon-terminal on MME, then cut and purify the intestinal kinase. Both MME and the amidation were verified by the standard mass spectrometry and two-stage, tandem mass spectrometry. Result The resultant gene fragment of the fusion protein was determined by PCR to be 837bp long as expected. In comparison to the theoretical value of 3 057.64, the relative molecular weight of MME obtained by the standard mass spectrometry was 3 057.7. The molecular weight of the amidated carbon-terminal fragments of MME was measured by the two-stage, tandem mass spectrometry to be 1,214.728, which was close to the known theoretical value of 1 214.739 with a matching rate of 45. It appeared that the carbon-terminal of MME had been effectively amidated, and the protein obtained was soluble. Conclusion The recombinant fusion proteins were successfully expressed in E. coli to enable the intein self-splitting in the presences of MESNa and NH₄HCO₃ and amidate the carbon terminal of MME. Therefore, the current simple, one-step preparation could achieve accurate, duplicatable, and meaningful results. By combining the standard mass spectrometry and two-stage, tandem mass spectrometry, the amidated carbon-terminal polypeptides could satisfactorily be detected.
- Antimicrobial peptides,
- amidation,
- intein mediation,
- self-split

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References(16)

References

[1]	LIN J G, XIA L Z, LIANG J X, et al. The roles of glucose metabolic reprogramming in chemo- and radio-resistance [J]. Journal of Experimental & Clinical Cancer Research, 2019, 38: 218.
[2]	Moravej H, Moravej Z, Yazdanparast M, et al. Antimicrobial peptides: features, action, and their resistance mechanisms in bacteria [J]. Microbial Drug Resistance, 2018, 24(6): 747−767. doi: 10.1089/mdr.2017.0392
[3]	YUAN Y P, ZAI Y, XI X P, et al. a novel membrane-disruptive antimicrobial peptide from frog skin secretion against cystic fibrosis isolates and evaluation of anti-MRSA effect using Galleria mellonella model [J]. Biochimica et Biophysica Acta. General Subjects, 2019, 1863(5): 849−856. doi: 10.1016/j.bbagen.2019.02.013
[4]	叶若柏, 吴珍红, 缪晓青. 具有抗肿瘤和抗菌活性的长效改性蜂毒肽(GPG)设计及其生物活性评价 [J]. 中国农学通报, 2020, 36(16):34−41. YE R B, WU Z H, MIAO X Q. Design and bioactivity evaluation of long-effective modified melittin (GPG) with antitumor and antibacterial function [J]. Chinese Agricultural Science Bulletin, 2020, 36(16): 34−41.(in Chinese)
[5]	祁丽, 姜宁, 张爱忠, 等. 抗菌肽多基因表达技术与策略 [J]. 黑龙江畜牧兽医(上半月), 2016(8):52−56. QI L, JIANG N, ZHANG A Z, et al. Multi-gene expression technology and strategy of antibacterial peptides [J]. Heilongjiang Animal Science and Veterinary Medicine, 2016(8): 52−56.(in Chinese)
[6]	QUINTERO-HERNÁNDEZ V, ORTIZ E, RENDÓN-ANAYA M, et al. Scorpion and spider venom peptides: Gene cloning and peptide expression [J]. Toxicon: Official Journal of the International Society on Toxinology, 2011, 58(8): 644−663. doi: 10.1016/j.toxicon.2011.09.015
[7]	ZHOU G, LI L, DENG W, et al. Intein-mediated recombinant expression of Cecropins AD, its amidation and application [J]. Journal of Parasitic Biology, 2016, 11(8):705−709.
[8]	FUHLENDORFF J, GETHER U, AAKERLUND L, et al. Leu31, Pro34]neuropeptide Y: A specific Y1 receptor agonist [J]. Proceedings of the National Academy of Sciences of the United States of America, 1990, 87(1): 182−186. doi: 10.1073/pnas.87.1.182
[9]	赵波. 酰胺化Exendin-4多肽的重组制备方法: CN1693459[P]. 2005-11-08.
[10]	RAY M V L, VAN DUYNE P, BERTELSENT A H, et al. Production of recombinant salmon calcitonin by in vitro amidation of an Escherichia coli produced precursor peptide [J]. Bio/Technology, 1993, 11(1): 64.
[11]	LI Y F. Split-inteins and their bioapplications [J]. Biotechnology Letters, 2015, 37(11): 2121−2137. doi: 10.1007/s10529-015-1905-2
[12]	俞超, 周冠, 李蕾, 等. 一种利用基因工程高效表达酰胺化EC07的方法: CN105274128A[P]. 2016-01-26.
[13]	STEVENS A J, BROWN Z Z, SHAH N H, et al. Design of a split intein with exceptional protein splicing activity [J]. Journal of the American Chemical Society, 2016, 138(7): 2162−2165. doi: 10.1021/jacs.5b13528
[14]	YANG Y H, CHEN H, YU C, et al. In vitro amidating processing of products expressed by gene engineering [J]. Acta Biochimica et Biophysica Sinica, 2000(3): 312−315.
[15]	ALBERTSEN L, SHAW A C, NORRILD J C, et al. Recombinant production of peptide C-terminal α-amides using an engineered intein [J]. Bioconjugate Chemistry, 2013, 24(11): 1883−1894. doi: 10.1021/bc4002689
[16]	江智红, 杨宇虹. 用毛细管电泳法进行C端酰胺化结构分析和酰胺化酶活性测定 [J]. 生物化学与生物物理学报:英文版, 1998, 30(1):21−25. JIANG Z H, YANG Y H. Analysis of C-terminal amidating structure and determination of amidating enzyme activity by using capillary electrophoresis [J]. Acta Biochimica et Biophysica Sinca, 1998, 30(1): 21−25.(in Chinese)