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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2023, Vol. 43 Issue (4): 30-40    DOI: 10.13523/j.cb.2210002
研究报告     
烟草NtASAT1基因的原核表达、纯化及功能验证*
韩丽1,**(),王丽娇1,肖成志1,董滋强1,杜悦1,何培新1,2
1.郑州轻工业大学食品与生物工程学院 郑州 450000
2.郑州轻工业大学食品生产与安全协同创新中心 郑州 450000
Prokaryotic Expression, Purification and Functional Verification of ASAT1 Gene of Nicotiana tabacum
HAN Li1,**(),WANG Li-jiao1,XIAO Cheng-zhi1,DONG Zi-qiang1,DU Yue1,HE Pei-xin1,2
1. School of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou 450000, China
2. Collaborative Innovation Center for Food Production and Safety, Zhengzhou University of Light Industry, Zhengzhou 450000, China
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摘要：

目的:烟草酰基糖酰基转移酶(Nicotiana tabacum acylsugar acyltransferase, NtASAT1)可催化短支链脂肪酸与蔗糖形成蔗糖单酯。利用大肠杆菌原核表达系统分析NtASAT1的表达,并对其进行纯化及功能验证。方法:首先利用生物信息学软件对烟草NtASAT1的理化性质、二级结构及同源性进行分析;之后从烟草腺毛的cDNA中克隆NtASAT1基因,并构建表达载体,研究其在BL21(DE3)中的表达情况;最后利用镍柱对NtASAT1进行纯化,将纯化后得到的目标蛋白进行酶反应,通过液相色谱-质谱法(liquid chromatography-mass spectrometry,LC-MS)检测产物并验证其功能活性。结果:C端截短93个氨基酸之后的NtASAT1能够在BL21(DE3)中表达。表达后的蛋白质大部分以不可溶状态存在于细胞中,不同的诱导剂浓度、诱导时间及诱导温度对于蛋白质可溶性表达影响不明显。利用镍柱对其纯化得到目标蛋白,加入底物进行酶反应,通过LC-MS检测到产物蔗糖单酯的存在。结论:通过克隆及纯化得到重组蛋白trNtASAT1,加入底物进行酶反应后可检测到产物,证明纯化后的NtASAT1具有一定功能,为ASAT类酶的纯化及应用奠定基础。
关键词： 烟草;  酰基糖酰基转移酶;  表达;  纯化    
Abstract:

Objective: It is reported that NtASAT1 (Nicotiana tabacum acylsugar acyltransferase) from tobacco can transform sucrose and short branched chain fatty acids to sucrose monoester. Thus, we tried to use the prokaryotic expression system of Escherichia coli to analyze the expression and purification condition of NtASAT1 and further verify the function of purified NtASAT1. Methods: First, the physical and chemical properties, secondary structure and homology of tobacco NtASAT1 were analyzed by bioinformatics software. Then, the gene NtASAT1 was cloned from the cDNA of tobacco glandular hairs and constructed into the expression vector so as to study its expression in BL21 (DE 3). Finally, NtASAT1 was purified by nickel column and the activity of purified target protein was then analyzed by enzyme reaction. The product of the enzymatic reaction was analyzed by liquid chromatography-mass spectrometry (LC-MS). Results: NtASAT1, which was truncated by 93 amino acids at the C-terminal, could be expressed in BL21 (DE3). Most of the expressed protein existed in an insoluble state and different concentrations of inducer, induction time and induction temperature had no obvious effect on the soluble expression of the protein trNtASAT1. The target protein was purified by nickel column and after enzyme reaction by adding substrate, the product sucrose monoester could be detected by LC-MS. Conclusion: The recombinant protein trNtASAT1 was cloned and purified and the enzymatic product sucrose monoester was detected by LC-MS, which proved that the purified NtASAT1 was functional. This study laid the foundation for purification and further application of enzyme ASAT in industry.
Key words: Nicotiana tabacum    Acylsugar acyltransferase    Expression    Purification
收稿日期: 2022-10-07 出版日期: 2023-05-04
ZTFLH:  Q786  
基金资助: 河南省重点研发与推广专项(202102310869)
通讯作者: **电子信箱:han_l@zzuli.edu.cn   
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韩丽
王丽娇
肖成志
董滋强
杜悦
何培新

引用本文:

韩丽, 王丽娇, 肖成志, 董滋强, 杜悦, 何培新. 烟草NtASAT1基因的原核表达、纯化及功能验证*[J]. 中国生物工程杂志, 2023, 43(4): 30-40.

HAN Li, WANG Li-jiao, XIAO Cheng-zhi, DONG Zi-qiang, DU Yue, HE Pei-xin. Prokaryotic Expression, Purification and Functional Verification of ASAT1 Gene of Nicotiana tabacum. China Biotechnology, 2023, 43(4): 30-40.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2210002    或    https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I4/30

图1  NtASAT1蛋白的二级结构
名称 核苷酸同源性/% 氨基酸同源性/%
SlASAT1 45.00 33.40
SlASAT2 44.48 31.10
SlASAT3 45.03 28.89
SlASAT4 38.89 20.83
PaxASAT1 66.92 60.11
PaxASAT2 45.18 33.83
PaxASAT3 47.13 35.06
PaxASAT4 42.74 28.85
SsASAT1 67.22 60.52
SsASAT2 43.77 35.38
SsASAT3 46.58 33.82
SsASAT5 38.84 30.80
表1  NtASAT1蛋白的同源性分析
图2  NtASAT1蛋白的进化树分析
图3  NtASAT1基因的克隆及表达载体的鉴定
图4  NtASAT1蛋白的表达
图5  不同诱导剂浓度、诱导时间、诱导温度对重组蛋白表达量的影响
图6  重组蛋白的纯化
图7  trNtASAT1酶活性验证
[1] Baker I J A, Furlong D N, Grieser F, et al. Sugar fatty acid ester surfactants: base-catalyzed hydrolysis. Journal of Surfactants and Detergents, 2000, 3(1): 29-32.
doi: 10.1007/s11743-000-0109-0
[2] de Sousa D P, Gonçalves J C R, Quintans-Júnior L, et al. Study of anticonvulsant effect of citronellol, a monoterpene alcohol, in rodents. Neuroscience Letters, 2006, 401(3): 231-235.
pmid: 16650577
[3] Ye R, Hayes D G. Lipase-catalyzed synthesis of saccharide-fatty acid esters utilizing solvent-free suspensions: effect of acyl donors and acceptors, and enzyme activity retention. Journal of the American Oil Chemists’ Society, 2012, 89(3): 455-463.
doi: 10.1007/s11746-011-1919-4
[4] Neta N S, Peres A M, Teixeira J A, et al. Maximization of fructose esters synthesis by response surface methodology. New Biotechnology, 2011, 28(4): 349-355.
doi: 10.1016/j.nbt.2011.02.007 pmid: 21356336
[5] 周莉, 刘波, 王玮, 等. 水乳化法合成蔗糖酯. 深圳大学学报, 1994, 11(S1): 87-91.
Zhou L, Liu B, Wang W, et al. Preparation of sucrose esters by emulsion method. Shenzhen University Journal, 1994, 11(S1): 87-91.
[6] 马亚茹. 糖酯的酶促合成及其功能特性的研究. 广州: 暨南大学, 2018.
Ma Y R. The enzymatic synthesis and functional properties of sugar esters. Guangzhou: Jinan University, 2018.
[7] 秦林莉, 刘郅骞, 张静, 等. 蔗糖酯在食品工业中的应用研究进展. 中国调味品, 2022, 47(4): 207-211, 220.
Qin L L, Liu Z Q, Zhang J, et al. Research progress on application of sucrose ester in food industry. China Condiment, 2022, 47(4): 207-211, 220.
[8] 施若晗, 陈敏, 张祥民. 二维色谱-质谱联用测定烟草中的蔗糖酯//中国化学会, 第22届全国色谱学术报告会及仪器展览会. 上海: 中国化学会, 2019: 364.
Shi R H, Chen M, Zhang X M. Determination of sucrose esters in tobacco by two-dimensional chromatography-mass spectrometry//Chinese Chemical Society, The 22nd National Chromatographic Symposium and Instrument Exhibition. Shanghai: Chinese Chemical Society, 2019: 364.
[9] 吴琼, 孙姣, 刘一宁, 等. 蔗糖酯在药物递送系统的应用研究进展. 精细化工, 2021, 38(2): 276-281.
Wu Q, Sun J, Liu Y N, et al. Research progress of application of sucrose esters in drug delivery system. Fine Chemicals, 2021, 38(2): 276-281.
[10] 曾清清, 张立彦. 鸡骨高汤乳化条件及乳化稳定性的研究. 中国调味品, 2014, 39(2): 1-6.
Zeng Q Q, Zhang L Y. Study on emulsification conditions and emulsion stability of chicken bone stock. China Condiment, 2014, 39(2): 1-6.
[11] 付莉, 宋更申. 药用辅料蔗糖硬脂酸酯的研究概况. 中国药师, 2013, 16(12): 1924-1925.
Fu L, Song G S. General situation of research on sucrose stearate as pharmaceutical auxiliary material. China Pharmacist, 2013, 16(12): 1924-1925.
[12] Chortyk O T, Pomonis J G, Johnson A W. Syntheses and characterizations of insecticidal sucrose esters. Journal of Agricultural and Food Chemistry, 1996, 44(6): 1551-1557.
doi: 10.1021/jf950615t
[13] Zheng Z Y, Qualley A, Fan B F, et al. An important role of a BAHD acyl transferase-like protein in plant innate immunity. The Plant Journal: for Cell and Molecular Biology, 2009, 57(6): 1040-1053.
doi: 10.1111/tpj.2009.57.issue-6
[14] Schilmiller A L, Charbonneau A L, Last R L. Identification of a BAHD acetyltransferase that produces protective acyl sugars in tomato trichomes. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(40): 16377-16382.
[15] St-Pierre B, De Luca V. Evolution of acyltransferase genes: origin and diversification of the BAHD superfamily of acyltransferases involved in secondary metabolism. Recent Advances in Phytochemistry, 2000, 34: 285-315.
[16] Nadakuduti S S, Uebler J B, Liu X X, et al. Characterization of trichome-expressed BAHD acyltransferases in Petunia axillaris reveals distinct acylsugar assembly mechanisms within the solanaceae. Plant Physiology, 2017, 175(1): 36-50.
doi: 10.1104/pp.17.00538 pmid: 28701351
[17] Chang A X, Hu Z Y, Chen B, et al. Characterization of trichome-specific BAHD acyltransferases involved in acylsugar biosynthesis in Nicotiana tabacum. Journal of Experimental Botany, 2022, 73(12): 3913-3928.
doi: 10.1093/jxb/erac095
[18] Fan P X, Miller A M, Schilmiller A L, et al. In vitro reconstruction and analysis of evolutionary variation of the tomato acylsucrose metabolic network. Proceedings of the National Academy of Sciences of the United States of America, 2016, 113(2): E239-E248.
[19] Schilmiller A L, Moghe G D, Fan P X, et al. Functionally divergent alleles and duplicated loci encoding an acyltransferase contribute to acylsugar metabolite diversity in Solanum trichomes. The Plant Cell, 2015, 27(4): 1002-1017.
doi: 10.1105/tpc.15.00087 pmid: 25862303
[20] Kim J, Kang K, Gonzales-Vigil E, et al. Striking natural diversity in glandular trichome acylsugar composition is shaped by variation at the acyltransferase 2 locus in the wild tomato Solanum habrochaites. Plant Physiology, 2012, 160(4): 1854-1870.
doi: 10.1104/pp.112.204735
[21] Moghe G D, Leong B J, Hurney S M, et al. Evolutionary routes to biochemical innovation revealed by integrative analysis of a plant-defense related specialized metabolic pathway. eLife, 2017, 6: e28468.
[22] 徐阳, 何好, 朱国庆, 等. 细叶百合LpPEX5基因克隆及蛋白表达纯化. 生物技术, 2020, 30(5): 417-423, 448.
Xu Y, He H, Zhu G Q, et al. Cloning of LpPEX5 gene from Lilium pumilum and protein expression and purification. Biotechnology, 2020, 30(5): 417-423, 448.
[23] 王会平, 郜赵伟, 刘冲, 等. CD14蛋白表达、纯化及单克隆抗体制备. 现代生物医学进展, 2020, 20(19): 3616-3620.
Wang H P, Gao Z W, Liu C, et al. Expression and purification of CD14 protein and preparation of anti-CD14 monoclonal antibody. Progress in Modern Biomedicine, 2020, 20(19): 3616-3620.
[24] 吴思思, 蒋维. 人生长分化因子11基因原核表达质粒的构建、表达及其蛋白的纯化. 中国生物制品学杂志, 2018, 31(7): 723-727.
Wu S S, Jiang W. Construction of prokaryotic expression vector for human growth differentiation factor 11 gene and expression and purification of target protein. Chinese Journal of Biologicals, 2018, 31(7): 723-727.
[25] 李聪, 田培洁, 张宇, 等. 烟草花叶病毒P54基因的原核表达与蛋白纯化. 福建农业学报, 2021, 36(2): 209-214.
Li C, Tian P J, Zhang Y, et al. Prokaryotic expression and purification of tobacco mosaic virus specific P54 protein. Fujian Journal of Agricultural Sciences, 2021, 36(2): 209-214.
[26] 刘雨雨, 莫婷, 王晓晖, 等. 植物来源BAHD酰基转移酶家族研究进展. 中国中药杂志, 2016, 41(12): 2175-2182.
Liu Y Y, Mo T, Wang X H, et al. Research progress of plant BAHD acyltransferase family. China Journal of Chinese Materia Medica, 2016, 41(12): 2175-2182.
[27] 苏鹏, 龚国利. 优化大肠杆菌表达外源蛋白的研究进展. 生物技术通报, 2017, 33(2): 16-23.
doi: 10.13560/j.cnki.biotech.bull.1985.2017.02.003
Su P, Gong G L. Research progress on optimizing the expression of exogenous proteins in Escherichia coli. Biotechnology Bulletin, 2017, 33(2): 16-23.
doi: 10.13560/j.cnki.biotech.bull.1985.2017.02.003
[28] Dhakal S, Sapkota K, Huang F Q, et al. Cloning, expression and purification of the low-complexity region of RanBP9 protein. Protein Expression and Purification, 2020, 172: 105630.
[29] 唐王刚, 司雨, 张娜, 等. 结核分枝杆菌蛋白Rv1872c的表达、纯化及其生物信息学分析. 中国生物制品学杂志, 2020, 33(10): 1117-1121, 1127.
Tang W G, Si Y, Zhang N, et al. Expression, purification and bioinformatics of Mycobacterium tuberculosis protein Rv1872c. Chinese Journal of Biologicals, 2020, 33(10): 1117-1121, 1127.
[30] 曹拓, 魏准, 彭湘明. 人CC趋化因子配体17重组蛋白的原核表达、纯化及趋化活性分析. 中国生物制品学杂志, 2020, 33(10): 1128-1133, 1142.
Cao T, Wei Z, Peng X M. Prokaryotic expression, purification and chemotactic activity of recombinant human CC chemokine ligand 17. Chinese Journal of Biologicals, 2020, 33(10): 1128-1133, 1142.
[31] 林丽淑, 龙韵洪, 徐丽惠, 等. 重组人抗缪勒管激素C-末端蛋白的原核表达及纯化. 中国生物制品学杂志, 2020, 33(8): 890-893.
Lin L S, Long Y H, Xu L H, et al. Prokaryotic expression and purification of recombinant C-terminal protein of human anti-Müllerian hormone. Chinese Journal of Biologicals, 2020, 33(8): 890-893.
[32] 梁秋瑾. SUMO蛋白酶Ulp1的固定化及其应用研究. 重庆: 西南大学, 2017.
Liang Q J. The study of the immobilization of Ulp1 protease and its applications. Chongqing: Southwest University, 2017.
[33] Puig O, Caspary F, Rigaut G, et al. The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods, 2001, 24(3): 218-229.
doi: 10.1006/meth.2001.1183 pmid: 11403571
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