基因工程表达内含肽介导的抗菌肽（MME）及其酰胺化

叶若柏; 吴珍红; 缪晓青

doi:10.19303/j.issn.1008-0384.2020.11.013

基因工程表达内含肽介导的抗菌肽（MME）及其酰胺化

doi: 10.19303/j.issn.1008-0384.2020.11.013

叶若柏^1,,
吴珍红²,
缪晓青^{1, 2, ,}

1.
福建农林大学食品科学学院，福建　福州　350002
2.
福建农林大学动物科学学院，福建　福州　350002

基金项目: 国家自然科学基金项目（51202030）

详细信息

作者简介:
叶若柏（1979−），男，讲师，硕士，在读博士生，研究方向：药物生物技术（ Email：354650728@qq.com）

通讯作者:
缪晓青（1959−），男，硕士，教授，博士生导师，研究方向：天然生物毒素提取、改性及利用研究（ E-mail：mxqsf88@126.com）

中图分类号: S 816.75
计量
- 文章访问数: 1115
- HTML全文浏览量: 98
- PDF下载量: 40
- 被引次数: 0
出版历程
- 收稿日期: 2020-10-10
- 修回日期: 2020-11-05
- 网络出版日期: 2021-01-26
- 刊出日期: 2020-11-30

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

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

摘要

摘要: 目的探讨在巯乙基磺酸钠（MESNa）与碳酸氢铵（NH₄HCO₃）存在下，对在大肠杆菌上表达的重组融合蛋白中内含肽（Intein）用硫醇（DTT）进行自剪切，促使抗菌肽（MME）碳末端实现酰胺化。方法构建用大肠杆菌表达内含肽介导的MME重组质粒，通过表达获得依次由“组氨酸、Sumo标签、MME、内含肽”构成的融合蛋白，用镍柱及透析进行纯化，在MESNa和NH₄HCO₃存在下，用 DTT对内含肽进行自剪切，促使MME碳末端酰胺化，肠激酶切割、纯化，得到碳末端酰胺化的MME。质谱和二级串联质谱配合使用，检测MME及其碳末端酰胺化。结果通过PCR，鉴定融合蛋白，其基因片段为837 bp，符合预期。质谱鉴定得MME相对分子量为3057.7与其理论值3057.64一致。二级串联质谱检测得MME碳端碎片的分子量为1214.728，与碳末端酰胺化的MME碳端碎片理论分子量1214.739相符，匹配度为45，表明本研究制备的MME碳末端已被酰胺化，该蛋白为可溶。结论用大肠杆菌成功地表达了重组融合蛋白，在MESNa与NH₄HCO₃存在下，促进了内含肽自剪切，MME碳末端被酰胺化，实现了简单的“一步法”制备。质谱与二级串联质谱配合使用，可灵敏、简单地对碳末端酰胺化多肽进行检测，可作为该项检测方法的一种选择，结果表明，本研究成功地制备了碳末端酰胺化的MME。
- 抗菌肽 /
- 酰胺化 /
- 内含肽介导 /
- 自剪切
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

HTML全文

图 1 融合蛋白基因片段PCR扩增结果

注：M：DNA标志物；1：PCR扩增的融合蛋白基因

Figure 1. Electrophoresis of PCR amplified products

Note: M: DNA marker; 1: PCR product of fusion protein.

下载: 全尺寸图片幻灯片

图 2 表达的融合蛋白SDS-PAGE

注：M：标样 1：沉淀；2：上清; 3：流出样;4：Buffer B清洗; 5：Buffer C清洗;6：Buffer D洗脱

Figure 2. SDS-PAGE for fusion protein

Note: M: protein marker; 1: precipitation; 2: supernatant; 3: efflux sample; 4: washing with Buffer B; 5: washing with Buffer C; 6: Buffer D elution.

下载: 全尺寸图片幻灯片

图 3 酰胺化的MME的纯化分析

注：M：标样；1：融合蛋白（纯化标签+MME）；2：酶切反应后样品；3：流出样。

Figure 3. SDS-PAGE for MME and purification analysis

Note: M: protein marker; 1: fusion protein (purification label+MME); 2: product after EK digestion; 3: efflux sample.

下载: 全尺寸图片幻灯片

图 4 表达产物的质谱图

Figure 4. MS of expression product

下载: 全尺寸图片幻灯片

图 5 2824号碎片离子流色谱图

注：K.AFPAVLKVLTTG.- +酰胺化（蛋白碳端末端）峰面积：1 846

Figure 5. HPLC of No.2824 fragment

Note: AFPAVLKVLTTG.-+Amidated (Protein C-term) peak area: 1 846.

下载: 全尺寸图片幻灯片

图 6 巯乙磺酸钠和碳酸氢铵存在下内含肽自剪切过程^[15]

注：Ⅰ：酰基转移；Ⅱ：内含肽转化为巯乙基磺酸根；Ⅲ：酰胺化；

Figure 6. Self-splitting process of intein in presences of MESNa and NH₄HCO₃^[15]

Note: RL27tm; purification label; Ⅰ: acyl transfer; Ⅱ: conversion of intein to 2-mercaptoethanesulfonate; Ⅲ: amidation.

下载: 全尺寸图片幻灯片

表 1 MME鉴定列表

Table 1. List of identified MME

碎片号 Query	质荷比 Observed	实验分子量 Mr（expt）	理论分子量 Mr（calc）	匹配度 Score	多肽的碎片序列 Peptide
175	373.235 6	744.456 6	744.453 4	39	K.AFPAVLK.V
524	405.216 2	808.417 8	808.415 3	54	R.LMEAFAK.R
599	413.211 7	824.408 8	824.410 2	63	R.LMEAFAK.R + Oxidation（M）
1091	442.236 6	882.458 6	882.459 9	41	R.FLYDGIR.I
1877	483.263 5	964.512 4	964.516 4	50	R.LMEAFAKR.Q
1879	483.264 8	964.515 0	964.516 4	20	R.RLMEAFAK.R
2368	355.177 6	1 062.511 0	1 062.513 0	49	R.QGKEMDSLR.F
2407	539.320 2	1 076.626 0	1 076.638 0	60	K.FGKAFPAVLK.V
2760	608.296 1	1 214.578 0	1 214.582 0	97	K.VSDGSSEIFFK.I
2824	608.371 4	1 214.728 0	1 214.739 0	45	K.AFPAVLKVLTTG.- + Amidated（Protein C-term）
2841	608.869 3	1 215.724 0	1 215.723 0	56	K.AFPAVLKVLTTG.-
3418	652.799 1	1 303.584 0	1 303.564 0	47	R.EQIGGDDDDKGR.G
6724	856.720 3	2 567.139 0	2 567.145 0	94	R.IQADQTPEDLDMEDNDIIEAHR.E
6741	862.046	2 583.116 0	2 583.140 0	61	R.IQADQTPEDLDMEDNDIIEAHR.E +Oxidation（M）

下载: 导出CSV

参考文献(16)

[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)