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
|
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.
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.
引文
[1]Yu K,Liu CC,Kim BG,et al.Synthetic fusion protein design and applications.Biotechnol Adv,2015,33(1):155-164.
[2]Kaur J,Kumar A,Kaur J.Strategies for optimization of heterologous protein expression in E.coli:roadblocks and reinforcements.Int J Biol Macromol2018,106:803-822.
[3]Zhang Q,Wu XY,Jiang XK,et al.Trend of hybrid enzyme design in the big data era.Chin J Biotech2018,34(7):1033-1045(in Chinese).张群,吴秀芸,蒋绪恺,等.大数据时代杂合酶的设计及其新趋势.生物工程学报,2018,34(7):1033-1045.
[4]Takano K,Okamoto T,Okada J,et al.Stabilization by fusion to the C-terminus of hyperthermophile Sulfolobus tokodaii RNase HI:a possibility of protein stabilization tag.PLoS ONE,2011,6(1):e16226.
[5]Lu XY,Liu S,Zhang DX,et al.Enhanced thermal stability and specific activity of Pseudomonas aeruginosa lipoxygenase by fusing with self-assembling amphipathic peptides.Appl Microbiol Biotechnol,2013,97(21):9419-9427.
[6]Zhao Q,Xu WH,Xing L,et al.Recombinant production of medium-to large-sized peptides in Escherichia coli using a cleavable self-aggregating tag.Microb Cell Fact,2016,15:136.
[7]Zhao WX,Liu S,Liu LM,et al.Analysis of the factors influencing the expression of enzymes fused with self-assembling amphipathic peptides.Food Ferment Ind,2017,43(12):1-6(in Chinese).赵伟欣,刘松,刘立明,等.自组装双亲短肽氨基酸组成及连接肽对其融合酶表达量的影响.食品与发酵工业,2017,43(12):1-6.
[8]Han RZ,Li JH,Shin HD,et al.Fusion of self-assembling amphipathic oligopeptides with cyclodextrin glycosyltransferase improves2-O-D-glucopyranosyl-L-ascorbic acid synthesis with soluble starch as the glycosyl donor.Appl Environ Microbiol,2014,80(15):4717-4724.
[9]Liu Y,Cui WJ,Liu ZM,et al.Enhancement of thermo-stability and product tolerance of Pseudomonas putida nitrile hydratase by fusing with self-assembling peptide.J Biosci Bioeng,2014,118(3):249-252.
[10]Yang HQ,Lu XY,Liu L,et al.Fusion of an oligopeptide to the N terminus of an alkalineα-amylase from Alkalimonas amylolytica simultaneously improves the enzyme’s catalytic efficiency,thermal stability,and resistance to oxidation.Appl Environ Microbiol,201379(9):3049-3058.
[11]Liu S,Wang MX,Du GC,et al.Improvement of thermal stability of alkaline polygalacturonate lyase by fusing with self-assembling amphipathic peptides.Food Ferment Ind,2015,41(11):1-6(in Chinese).刘松,汪明星,堵国成,等.融合自组装双亲短肽提高碱性果胶酶热稳定性.食品与发酵工业,2015,41(11):1-6.
[12]Zhao WX,Liu LM,Du GC,et al.A multifunctional tag with the ability to benefit the expression,purification,thermostability and activity of recombinant proteins.J Biotechnol,2018,283:1-10.
[13]Goodman DB,Church GM,Kosuri S.Causes and effects of N-Terminal codon bias in bacterial genes.Science,2013,342(6157):475-479.
[14]Zhong C,Wei P,Zhang YHP.Enhancing functional expression of codon-optimized heterologous enzymes in Escherichia coli BL21(DE3)by selective introduction of synonymous rare codons.Biotechnol Bioeng,2017,114(5):1054-1064.
[15]Chen X.Study on the expression and fermentation optimization of L-asparaginase in Bacillus subtilis WB600[D].Wuxi:Jiangnan University,2015(in Chinese).陈璇.L-天冬酰胺酶在Bacillus subtilis WB600中的表达与发酵过程优化研究[D].无锡:江南大学,2015.
[16]Ren CH,Zhang J,Du GC,et al.Enhancing thermal stability of glucose oxidase by fusing amphiphilic short peptide.Chin J Biotech,2018,34(7):1106-1116(in Chinese).任春慧,张娟,堵国成,等.基于融合双亲短肽提高葡萄糖氧化酶的热稳定性.生物工程学报,2018,34(7):1106-1116.
[17]Wang MX,Liu S,Liu L,et al.Fusions of amphipathic peptide to the alkaline polygalacturonate lyase from Bacillus sp.WSHB04-02 improves the production.J Food Sci Biotechnol,2016,35(5):504-509(in Chinese).汪明星,刘松,刘龙,等.融合短肽促进碱性果胶酶的高效表达.食品与生物技术学报,2016,35(5):504-509.
[18]Qiu FF.Study on the high-level expression and thermal stability of Pseudomonas aeruginosa lipoxygenase[D].Wuxi:Jiangnan University,2017(in Chinese).邱芳芳.重组脂肪氧合酶的高效表达和热稳定性研究[D].无锡:江南大学,2017.
[19]Lu XY,Zhang J,Liu S,et al.Overproduction purification,and characterization of extracellular lipoxygenase of Pseudomonas aeruginosa in Escherichia coli.Appl Microbiol Biotechnol,2013,97(13):5793-5800.
[20]O'Fágáin C.Enzyme stabilization-recent experimental progress.Enzyme Microb Technol,2003,33(2/3):137-149.
[21]Charneski CA,Hurst LD.Positively charged residues are the major determinants of ribosomal velocity PLoS Biol,2013,11(3):e1001508.
[22]Mei YZ,Chen YR,Zhai RY,et al.Cloning purification and biochemical properties of a thermostable pectinase from Bacillus halodurans M29.J Mol Catal B:Enzymat,2013,94:77-81.
[23]Kosobokova EN,Skrypnik KA,Kosorukov VS.Overview of fusion tags for recombinant proteins.Biochemistry(Moscow),2016,81(3):187-200.
[24]Lin ZL,Zhou BH,Wu W,et al.Self-assembling amphipathic alpha-helical peptides induce the formation of active protein aggregates in vivo Faraday Dis,2013,166:243-256.
[25]Becker K,Van Alstine J,Bülow L.Multipurpose peptide tags for protein isolation.J Chromatogr A2008,1202(1):40-46.
[26]Farchaus JW,Ribot WJ,Jendrek S,et al.Fermentation,purification,and characterization of protective antigen from a recombinant,avirulent strain of Bacillus anthracis.Appl Environ Microbiol,1998,64(3):982-991.
[2]Kaur J,Kumar A,Kaur J.Strategies for optimization of heterologous protein expression in E.coli:roadblocks and reinforcements.Int J Biol Macromol2018,106:803-822.
[3]Zhang Q,Wu XY,Jiang XK,et al.Trend of hybrid enzyme design in the big data era.Chin J Biotech2018,34(7):1033-1045(in Chinese).张群,吴秀芸,蒋绪恺,等.大数据时代杂合酶的设计及其新趋势.生物工程学报,2018,34(7):1033-1045.
[4]Takano K,Okamoto T,Okada J,et al.Stabilization by fusion to the C-terminus of hyperthermophile Sulfolobus tokodaii RNase HI:a possibility of protein stabilization tag.PLoS ONE,2011,6(1):e16226.
[5]Lu XY,Liu S,Zhang DX,et al.Enhanced thermal stability and specific activity of Pseudomonas aeruginosa lipoxygenase by fusing with self-assembling amphipathic peptides.Appl Microbiol Biotechnol,2013,97(21):9419-9427.
[6]Zhao Q,Xu WH,Xing L,et al.Recombinant production of medium-to large-sized peptides in Escherichia coli using a cleavable self-aggregating tag.Microb Cell Fact,2016,15:136.
[7]Zhao WX,Liu S,Liu LM,et al.Analysis of the factors influencing the expression of enzymes fused with self-assembling amphipathic peptides.Food Ferment Ind,2017,43(12):1-6(in Chinese).赵伟欣,刘松,刘立明,等.自组装双亲短肽氨基酸组成及连接肽对其融合酶表达量的影响.食品与发酵工业,2017,43(12):1-6.
[8]Han RZ,Li JH,Shin HD,et al.Fusion of self-assembling amphipathic oligopeptides with cyclodextrin glycosyltransferase improves2-O-D-glucopyranosyl-L-ascorbic acid synthesis with soluble starch as the glycosyl donor.Appl Environ Microbiol,2014,80(15):4717-4724.
[9]Liu Y,Cui WJ,Liu ZM,et al.Enhancement of thermo-stability and product tolerance of Pseudomonas putida nitrile hydratase by fusing with self-assembling peptide.J Biosci Bioeng,2014,118(3):249-252.
[10]Yang HQ,Lu XY,Liu L,et al.Fusion of an oligopeptide to the N terminus of an alkalineα-amylase from Alkalimonas amylolytica simultaneously improves the enzyme’s catalytic efficiency,thermal stability,and resistance to oxidation.Appl Environ Microbiol,201379(9):3049-3058.
[11]Liu S,Wang MX,Du GC,et al.Improvement of thermal stability of alkaline polygalacturonate lyase by fusing with self-assembling amphipathic peptides.Food Ferment Ind,2015,41(11):1-6(in Chinese).刘松,汪明星,堵国成,等.融合自组装双亲短肽提高碱性果胶酶热稳定性.食品与发酵工业,2015,41(11):1-6.
[12]Zhao WX,Liu LM,Du GC,et al.A multifunctional tag with the ability to benefit the expression,purification,thermostability and activity of recombinant proteins.J Biotechnol,2018,283:1-10.
[13]Goodman DB,Church GM,Kosuri S.Causes and effects of N-Terminal codon bias in bacterial genes.Science,2013,342(6157):475-479.
[14]Zhong C,Wei P,Zhang YHP.Enhancing functional expression of codon-optimized heterologous enzymes in Escherichia coli BL21(DE3)by selective introduction of synonymous rare codons.Biotechnol Bioeng,2017,114(5):1054-1064.
[15]Chen X.Study on the expression and fermentation optimization of L-asparaginase in Bacillus subtilis WB600[D].Wuxi:Jiangnan University,2015(in Chinese).陈璇.L-天冬酰胺酶在Bacillus subtilis WB600中的表达与发酵过程优化研究[D].无锡:江南大学,2015.
[16]Ren CH,Zhang J,Du GC,et al.Enhancing thermal stability of glucose oxidase by fusing amphiphilic short peptide.Chin J Biotech,2018,34(7):1106-1116(in Chinese).任春慧,张娟,堵国成,等.基于融合双亲短肽提高葡萄糖氧化酶的热稳定性.生物工程学报,2018,34(7):1106-1116.
[17]Wang MX,Liu S,Liu L,et al.Fusions of amphipathic peptide to the alkaline polygalacturonate lyase from Bacillus sp.WSHB04-02 improves the production.J Food Sci Biotechnol,2016,35(5):504-509(in Chinese).汪明星,刘松,刘龙,等.融合短肽促进碱性果胶酶的高效表达.食品与生物技术学报,2016,35(5):504-509.
[18]Qiu FF.Study on the high-level expression and thermal stability of Pseudomonas aeruginosa lipoxygenase[D].Wuxi:Jiangnan University,2017(in Chinese).邱芳芳.重组脂肪氧合酶的高效表达和热稳定性研究[D].无锡:江南大学,2017.
[19]Lu XY,Zhang J,Liu S,et al.Overproduction purification,and characterization of extracellular lipoxygenase of Pseudomonas aeruginosa in Escherichia coli.Appl Microbiol Biotechnol,2013,97(13):5793-5800.
[20]O'Fágáin C.Enzyme stabilization-recent experimental progress.Enzyme Microb Technol,2003,33(2/3):137-149.
[21]Charneski CA,Hurst LD.Positively charged residues are the major determinants of ribosomal velocity PLoS Biol,2013,11(3):e1001508.
[22]Mei YZ,Chen YR,Zhai RY,et al.Cloning purification and biochemical properties of a thermostable pectinase from Bacillus halodurans M29.J Mol Catal B:Enzymat,2013,94:77-81.
[23]Kosobokova EN,Skrypnik KA,Kosorukov VS.Overview of fusion tags for recombinant proteins.Biochemistry(Moscow),2016,81(3):187-200.
[24]Lin ZL,Zhou BH,Wu W,et al.Self-assembling amphipathic alpha-helical peptides induce the formation of active protein aggregates in vivo Faraday Dis,2013,166:243-256.
[25]Becker K,Van Alstine J,Bülow L.Multipurpose peptide tags for protein isolation.J Chromatogr A2008,1202(1):40-46.
[26]Farchaus JW,Ribot WJ,Jendrek S,et al.Fermentation,purification,and characterization of protective antigen from a recombinant,avirulent strain of Bacillus anthracis.Appl Environ Microbiol,1998,64(3):982-991.