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Volume 35 Issue 11
Nov.  2020
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Article Contents
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
Citation: 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

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

doi: 10.19303/j.issn.1008-0384.2020.11.013
  • Received Date: 2020-10-10
  • Rev Recd Date: 2020-11-05
  • Available Online: 2021-01-26
  • Publish Date: 2020-11-28
  •   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.
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  • [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)
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