组氨酸标签蛋白纯化介质的合成及其分离纯化
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摘要
蛋白质的分离纯化是发展蛋白组学,研究蛋白质结构功能与应用的前提和基础。本文研发了一种基于亲和性离子液体(AIL)/离子液体(IL)的组氨酸标签蛋白的纯化体系,经螯合金属离子(Cu~(2+)、Zn~(2+)、Ni~(2+))后应用于从大肠杆菌细胞裂解物中纯化组氨酸标签增强型绿色荧光蛋白。结果表明:Cu~(2+)-AIL/IL对目标蛋白的亲和性能最高,为62%,然而含有较多的杂蛋白成分;Zn~(2+)-AIL/IL的亲和效率约为51%,且纯化后目标蛋白的纯度高达90%,Ni~(2+)-AIL/IL的亲和能力最弱。进一步将Zn~(2+)-AIL/IL体系应用于直接纯化大肠杆菌中表达的组氨酸标签青霉素结合蛋白的细菌裂解液,所得目标蛋白纯度高达95%。这种方法为新型组氨酸标签蛋白纯化介质,从基础研究拓展到产业化应用提供了理论依据,并可望将此类新型亲和介质应用到其它蛋白质的分离纯化。
The isolation and purification of a protein is an essential first step before detailed functional and structural characterization studies can commence. This article reported a His-tagged protein purification system based on affinity ionic liquids(AIL)/ionic liquid(IL) and chelated metal ion(Cu, Zn, Ni), which was successfully applied to the purification of histidine-labelled enhanced green fluorescent protein(His-tagged EGFP) from cell lysate of E. coli. The results indicated that: Cu~(2+)-AIL/IL showed the best affinity with the targeted protein but giving a low purity, and Zn~(2+)-AIL/IL was proven to be the best affinity ionic liquid by which 51% of the total target protein was recovered with purity of 90%, while the affinity of Ni~(2+)-AIL/IL was weakest among them. We further utilized Zn~(2+)-AIL/IL system to directly purify His-tagged penicillin binding protein expressed in E. coli., obtaining the targeted protein with a purity of as high as 95%. This method established a foundation for the separation and purification of His-tagged proteins from basic research to industrialization. The presented affinity medium can be further applied to the separation and purification of the other proteins from genetic engineering bacteria.
The isolation and purification of a protein is an essential first step before detailed functional and structural characterization studies can commence. This article reported a His-tagged protein purification system based on affinity ionic liquids(AIL)/ionic liquid(IL) and chelated metal ion(Cu, Zn, Ni), which was successfully applied to the purification of histidine-labelled enhanced green fluorescent protein(His-tagged EGFP) from cell lysate of E. coli. The results indicated that: Cu~(2+)-AIL/IL showed the best affinity with the targeted protein but giving a low purity, and Zn~(2+)-AIL/IL was proven to be the best affinity ionic liquid by which 51% of the total target protein was recovered with purity of 90%, while the affinity of Ni~(2+)-AIL/IL was weakest among them. We further utilized Zn~(2+)-AIL/IL system to directly purify His-tagged penicillin binding protein expressed in E. coli., obtaining the targeted protein with a purity of as high as 95%. This method established a foundation for the separation and purification of His-tagged proteins from basic research to industrialization. The presented affinity medium can be further applied to the separation and purification of the other proteins from genetic engineering bacteria.
引文
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[16]XU W Y,CAO H Z,REN G W,et al.An AIL/IL-based liquid/liquid extraction system for the purification of His-tagged proteins[J].Applied Microbiology and Biotechnology,2014,98(12):5665-5675.
[17]SPRATT B G,PARDEE A B.Penicillin-binding proteins and cell shape in E.coli[J].Nature,1975,254(5500):516-517.
[18]KOCAOGLU O,CALVO R A,SHAM L T,et al.Selective penicillin-binding protein imaging probes reveal substructure in bacterial cell division[J].ACS Chemical Biology,2012,7(10):1746-1753.
[19]SAUVAGE E,DEROUAUX A,FRAIPONT C,et al.Crystal structure of penicillin-binding protein 3(PBP3)from Escherichia coli[J].Plos One,2014,9(5):e98042.
[2]刘志国.基因工程原理与技术[M].北京:化学工业出版社,2011:265-266.
[3]MOONEY J T,FREDERICKS D,HEARN M T.Use of phage display methods to identify heptapeptide sequences for use as affinity purification‘tags’with novel chelating ligands in immobilized metal ion affinity chromatography[J].Journal of Chromatography A,2011,1218(1):92-99.
[4]PORATH J,CARLSSON J,OLSSON I,et al.Metal chelate affinity chromatography,a new approach to protein fractionation[J].Nature,1975,258(5536):598-599.
[5]CHEUNG R C F,WONG J H,NG T B.Immobilized metal ion affinity chromatography:a review on its applications[J].Applied Microbiology and Biotechnology,2012,96(6):1411-1420.
[6]SHENG C Z,HAN J,WANG Y,et al.Liquid liquid equilibria of ionic liquid 1-(2-methoxyethyl)-3-methylimidazolium bromide plus potassium carbonate,potassium phosphate,dipotassium phosphate plus water aqueous twophase systems[J].Fluid Phase Equilibria,2014,364:55-61.
[7]CLAUDIO A F M,MARQUES C F C,BOAL-PALHEIROS I,et al.Development of back-extraction and recyclability routes for ionic-liquid-based aqueous two-phase systems[J].Green Chemistry,2014,16(1):259-268.
[8]NEVES C M S S,KURNIA K A,SHIMIZU K,et al.The impact of ionic liquid fluorinated moieties on their thermophysical properties and aqueous phase behavior[J].Physical Chemistry Chemical Physics,2014,16(39):21340-21348.
[9]LIN X,WANG Y Z,ZENG Q,et al.Extraction and separation of proteins by ionic liquid aqueous two-phase system[J].Analyst,2013,138(21):6445-6453.
[10]NOORASHIKIN M S,MOHAMAD S,ABAS M R.Extraction and determination of parabens in water samples using an aqueous two-phase system of ionic liquid and salts with beta-cyclodextrin as the modifier coupled with high performance liquid chromatography[J].Analytical Methods,2014,6(2):419-425.
[11]MAREK A,KULHANEK J,LUDWIG M,et al.Facile synthesis of optically active imidazole derivatives[J].Molecules,2007,12(5):1183-90.
[12]LIANG X Z,YANG J G.Synthesis of a novel multi-SO3H functionalized ionic liquid and its catalytic activities for biodiesel synthesis[J].Green Chemistry,2010,12(2):201-204.
[13]ATTRI P,VENKATESU P,KUMAR A.Water and a protic ionic liquid acted as refolding additives for chemically denatured enzymes[J].Organic&Biomolecular Chemistry,2012,10(37):7475-7478.
[14]LAN Q,BASSI A S,ZHU J X J,et al.A modified Langmuir model for the prediction of the effects of ionic strength on the equilibrium characteristics of protein adsorption onto ion exchange/affinity adsorbents[J].Chemical Engineering Journal,2001,81(1):179-186.
[15]RAGHURAMAN H,GANGULY S,CHATTOPADHYAY A.Effect of ionic strength on the organization and dynamics of membrane-bound melittin[J].Biophysical Chemistry,2006,124(2):115-24.
[16]XU W Y,CAO H Z,REN G W,et al.An AIL/IL-based liquid/liquid extraction system for the purification of His-tagged proteins[J].Applied Microbiology and Biotechnology,2014,98(12):5665-5675.
[17]SPRATT B G,PARDEE A B.Penicillin-binding proteins and cell shape in E.coli[J].Nature,1975,254(5500):516-517.
[18]KOCAOGLU O,CALVO R A,SHAM L T,et al.Selective penicillin-binding protein imaging probes reveal substructure in bacterial cell division[J].ACS Chemical Biology,2012,7(10):1746-1753.
[19]SAUVAGE E,DEROUAUX A,FRAIPONT C,et al.Crystal structure of penicillin-binding protein 3(PBP3)from Escherichia coli[J].Plos One,2014,9(5):e98042.