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一种可促进重组蛋白表达量和稳定性的多功能纯化标签的开发与利用 被引量:1

Development of a purification tag to produce thermostable fused protein
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摘要 自组装双亲短肽(Self-assembling amphipathic peptides,SAPs)是一类亲疏水氨基酸按一定规律分布、具有自聚合效应的短肽,融合在酶蛋白N端时,具有促进表达和稳定化的功能。根据前期研究结论,设计一条全新的基于SAPs (S1vw,HNANARARHNANARARHNANARARHNARARAR)的可促进融合蛋白表达和稳定性,并可用于镍柱亲和层析的多功能短肽标签,在大肠杆菌表达系统中,将S1vw以PT-linker(PTPPTTPTPPTTPTP)融合在碱性果胶酶(Alkaline polygalacturonate lyase,PGL)、脂肪氧合酶(Lipoxygenase,LOX)及绿色荧光蛋白(Green fluorescent protein,GFP)的N末端时,与对应的野生型相比,PGL及LOX的粗酶活分别提高了3.1倍和1.89倍,GFP的荧光强度提高了16.22倍。S1vw的3种融合酶均可用镍柱进行亲和纯化,并具有较高的回收率。PGL及LOX在对应的热处理条件下,与野生型相比,半衰期分别提高了2.16倍和3.2倍。将GFP-S1vw在枯草芽孢杆菌及毕赤酵母表达系统中表达,发现在枯草芽孢杆菌中融合蛋白表达量提高明显,但在毕赤酵母中表达量几乎没有改变。说明在原核表达体系中,S1vw可作为一种新型的促表达、稳定化及可纯化的多功能标签。 Self-assembling amphipathic peptides(SAPs)have alternating hydrophilic and hydrophobic residues and can affect the thermal stabilities and catalytic properties of the fused enzymes.In this study,a novel multifunctional tag,S1vw(HNANARARHNANARARHNANARARHNARARAR)was developed to modify fused enzymes.After fusing S1vw at the enzymes/proteins N-terminus through a PT-linker,the crude enzymatic activities of polygalacturonate lyase and lipoxygenase were enhanced 3.1-and 1.89-fold,respectively,compared to the wild-type proteins.The relative fluorescence intensity of the green fluorescent protein was enhanced 16.22-fold.All the three S1vw fusions could be purified by nickel column with high purities and acceptable recovery rates.Moreover,S1vw also induced the thermostabilities enhancement of the fusions,with polygalacturonate lyase and lipoxygenase fusions exhibiting 2.16-and 3.2-fold increase compared with the corresponding wild-type,respectively.In addition,S1vw could enhance the production yield of green fluorescent protein in Escherichia coli and Bacillus subtilis while the production of GFP and its S1vw fusion changed slightly in Pichia pastoris.These results indicated that S1vw could be used as a multifunctional tag to benefit the production,thermal stability and purification of the fusion protein in prokaryotic expression system.
作者 赵伟欣 刘松 刘立明 陈坚 堵国成 Weixin Zhao;Song Liu;Liming Liu;Jian Chen;Guocheng Du(School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China)
出处 《生物工程学报》 CAS CSCD 北大核心 2019年第4期626-635,共10页 Chinese Journal of Biotechnology
基金 国家自然科学基金(No.31401638)资助~~
关键词 自组装双亲短肽 融合蛋白 表达量 热稳定性 纯化 表达宿主 self-assembling amphipathic peptides fusion enzymes production yield thermal stability purification expression host
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  • 1黄俊丽,李常军,王贵学.微生物果胶酶的分子生物学及其应用研究进展[J].生物技术通讯,2006,17(6):992-994. 被引量:17
  • 2WANG Y,WANG Z,DU G,et al.Enhancement of alkaline polygalacturonate lyase production in recombinant Pichia pastoris according to the ratio of methanol to cell concentration[J].Bioresource Technology,2009,100(3):1 343-1349.
  • 3Kashyap D R,Vohra P K,Chopra S,et al.Applications of pectinases in the commercial sector:a review[J].Bioresource Technology,2001,77(3):215-27.
  • 4Reid I,Ricard M.Pectinase in papermaking:solving retention problems in mechanical pulps bleached with hydrogen peroxide[J].Enzyme and Microbial Technology,2000,26(2-4):115-123.
  • 5Berensmeier S,Singh S A,Meens J,et al.Cloning of the pel A gene from Bacillus licheniformis 14A and biochemical characterization of recombinant,thermostable,high-alkaline pectate lyase[J].Applied Microbiology and Biotechnology,2004,64(4):560-567.
  • 6Kluskens L D,van Alebeek G J W M,Voragen A G J,et al.Molecular and biochemical characterization of the thermoactive family 1 pectate lyase from the hyperthermophilic bacterium Thermotoga maritima[J].Biochemical Journal,2003,370(2):651-659.
  • 7Hatada Y,Kobayashi T,Ito S.Enzymatic properties of the highly thermophilic and alkaline pectate lyase Pel-4B from alkaliphilic Bacillus sp strain P-4-N and the entire nucleotide and amino acid sequences[J].Extremophiles,2001,5(2):127-133.
  • 8Bommarius A S,Blum J K,Abrahamson M J.Status of protein engineering for biocatalysts:how to design an industrially useful biocatalyst[J].Current Opinion in Chemical Biology,2011,15(2):194-200.
  • 9ZHANG S,Lockshin C,Herbert A,et al.Zuotin,a putative Z-DNA binding protein in Saccharomyces cerevisiae[J].The EMBO Journal,1992,11(10):3 787.
  • 10LU X Y,LIU S,ZHANG D X,et al.Enhanced thermal stability and specific activity of Pseudomonas aeruginosa lipoxygenase by fusing with self-assembling amphipathic peptides[J].Applied Microbiology and Biotechnology,2013,97(21):9 419-9 427.

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