人心肌型脂肪酸结合蛋白在大肠杆菌中的可溶性表达和纯化
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摘要
人心脏型脂肪酸结合蛋白(Heart type-fatty acid binding protein,H-FABP)是存在于心肌细胞质内的小分子可溶性蛋白质,占心脏全部可溶性蛋白的4%~8%,具有很强的器官特异性。在急性心肌梗塞(AMI)早期,心肌细胞中H-FABP大量增加,因此可将其作为急性心肌梗塞等心肌损伤的生化标志物。大肠杆菌是常用的可快速生产重组蛋白的宿主,且融合或嵌合"标签"蛋白连接靶蛋白不仅能提高靶蛋白的溶解性,同时也可以防止细胞内的蛋白水解。作者旨在获得重组可溶性人心肌型脂肪酸结合蛋白(H-FABP),为进一步开发应用奠定基础。选择麦芽糖结合蛋白(MBP)作为H-FABP的融合标签蛋白,以提高H-FABP在大肠杆菌中的可溶性表达能力。通过RT-PCR扩增人H-FABP的基因序列,将目的基因克隆入pET28a表达载体,转化到DH5α感受态细菌中扩增得到原核表达重组质粒pET28a-MBP-H-FABP。将质粒转化入大肠杆菌BL21中,通过IPTG诱导,表达MBPH-FABP融合蛋白,同时SDS-PAGE分析验证。后经镍固定金属亲和层析纯化及离子亲和纯化法纯化,并经SDSPAGE和免疫印迹分析鉴定纯化后重组蛋白的免疫特异性。SDS-PAGE和免疫印迹分析显示重组蛋白相对分子质量约为55 k。经Ni~(+2)-NTA纯化后,MBP-H-FABP重组蛋白在SDS-PAGE电泳下获得单一条带,且MBP-H-FABP可与商业化的H-FABP单克隆抗体(Clone 12-1)呈特异性反应。其意义在于提供在大肠杆菌中制备可溶性重组人H-FABP的方法,为研究其生物学功能和制备单克隆抗体以及今后进行大规模的生产奠定了基础。
Heart type-fatty acid binding protein(H-FABP),a small molecule soluble protein that exists in the cytoplasm of the heart muscle,accounting for 4% ~ 8% of the total soluble protein of the heart,with strong organ specificity. In the early stage of acute myocardial infarction(AMI),the H-FABP in myocardial cells increased greatly,so it could be used as a biochemical marker for myocardial injury such as AMI. E. coli is a common host for quick production of recombinant proteins,and fusion or chimeric‘tags' protein can not only improve the solubility of target protein,but also prevent cell protein hydrolysis. In this study,we aimed to obtain soluble recombinant human heart type fatty acid binding protein(H-FABP) by prokaryotic expression for clinical use in diagnosis. Choosing maltose binding protein(MBP) as fusion label protein,in order to improve H-FABP soluble expression in E. coli.The encoding sequence of human H-FABP was amplified with RT-PCR and cloned into plasmid pET28 a,which then transformed into DH5α,to establish the prokaryotic expressing system. E. coli BL21 was transformed by the recombinant plasmid p ET28 a-MBP-HFABP. Expression of the recombinant protein was induced with IPTG. For scaled production,we purified the MBP-H-FABP fused protein by using immobilized metal affinity chromatography(Ni~(+2)-NTA agarose column) and cation-affinity purification,and then assayed recombinant products by SDS-PAGE and immunoblot. The molecular weight of the expressed fusion-protein was 55 k in SDSPAGE as expected. After Ni~+-NTA agarose column,we obtained purified recombinant protein at a single band by SDS-PAGE electrophoresis. And MBP-H-FABP fused protein could react specifically with commercial H-FABP polyclonal antibody(Clone 12-1). We provide a method to produce large amounts of soluble recombinant human H-FABP in E. coli,which may benefit for preparation of specific antibodies and diagnosis for some related diseases and lay a foundation for large-scale production in the future.
Heart type-fatty acid binding protein(H-FABP),a small molecule soluble protein that exists in the cytoplasm of the heart muscle,accounting for 4% ~ 8% of the total soluble protein of the heart,with strong organ specificity. In the early stage of acute myocardial infarction(AMI),the H-FABP in myocardial cells increased greatly,so it could be used as a biochemical marker for myocardial injury such as AMI. E. coli is a common host for quick production of recombinant proteins,and fusion or chimeric‘tags' protein can not only improve the solubility of target protein,but also prevent cell protein hydrolysis. In this study,we aimed to obtain soluble recombinant human heart type fatty acid binding protein(H-FABP) by prokaryotic expression for clinical use in diagnosis. Choosing maltose binding protein(MBP) as fusion label protein,in order to improve H-FABP soluble expression in E. coli.The encoding sequence of human H-FABP was amplified with RT-PCR and cloned into plasmid pET28 a,which then transformed into DH5α,to establish the prokaryotic expressing system. E. coli BL21 was transformed by the recombinant plasmid p ET28 a-MBP-HFABP. Expression of the recombinant protein was induced with IPTG. For scaled production,we purified the MBP-H-FABP fused protein by using immobilized metal affinity chromatography(Ni~(+2)-NTA agarose column) and cation-affinity purification,and then assayed recombinant products by SDS-PAGE and immunoblot. The molecular weight of the expressed fusion-protein was 55 k in SDSPAGE as expected. After Ni~+-NTA agarose column,we obtained purified recombinant protein at a single band by SDS-PAGE electrophoresis. And MBP-H-FABP fused protein could react specifically with commercial H-FABP polyclonal antibody(Clone 12-1). We provide a method to produce large amounts of soluble recombinant human H-FABP in E. coli,which may benefit for preparation of specific antibodies and diagnosis for some related diseases and lay a foundation for large-scale production in the future.
引文
[1]Roos W,Eymann E,Symannek M,et al.Monoclonal antibodies to human heart fatty acid-binding protein[J].J Immunol Med,1995,183(1):149-153.
[2]Tanaka T,Hirota Y,Sohmiya K,et al.Serum and urinary human heart fatty acid-binding protein in acute myocardial infarction[J].Clin Biochem,1991,24:195.
[3]Knowlton AA,Burrier RE,Brecher P.Rabbit heart fatty acidbinding protein[J].Circul Res,1989,65:981.
[4]Gorski J,Hermens W T,Borawski J.Increased fatty acid-binding protein concentration in plasma of patients with chronic ranal failure[J].Clin Chem,1997,43:193.
[5]Keagten JA,Nieuweuhoven FAV,Dieijen Visser MPV,et al.Distribation of myoglobin and fatty acid-binding protein in human cardiac autopsises[J].Clin Chem,1996,42:337.
[6]Glatz JF,Vander Vusse GJ,Simoons ML,et al.Fatty acid-binding protein and the early detection of acute myocardial infarction[J].Clin Chim Acta,1998,272:87.
[7]Alansari SE,Croal BL.Diagnostic value of heart fatty acid binding protein and myoglobin in patients admitted with chest pain[J].Ann Clin Biochem,2004,41:391.
[8]Koichi S,Yoshihiko S,Takeshi,et al.Ongoing myocardial damage in chronic heart failure is related to activated tumor necrosis factor and fas/fas ligand system[J].Circulation J,2004,68:747.
[9]Sorensen HP,Mortensen KK.Advanced genetic strategies for recombinant protein expression in Escherichia coli[J].J Biotechnol,2005,115(2):113-128.
[10]Davis GD,Elisee C,Newham DM,et al.New fusion protein systems designed to give soluble expression in Escherichia coli[J].Biotechnol Bioeng,1999,65(4):382-388.
[11]Sorensen HP,Sperling-Petersen HU,Mortensen KK.A favorable solubility partner for the recombinant expression of streptavidin[J].Protein Express Purific,2003,32(2):252-259.
[12]Jacquet A,Daminet V,Haumont M,et al.Expression of a recombinant Toxoplasma gondii ROP2 fragment as a fusion protein in bacteria circumvents insolubility and proteolytic degradation[J].Protein Express Purific,1999,17(3):392-400.
[13]丛珊,张阳,张竹君,等.心脏型脂肪酸结合蛋白(H-FABP)原核表达、纯化及多克隆抗体制备研究[J].中国医药指南,2012,4:1-4.
[14]Schaap F,Specht B,Ger J,et al.One-step purification of rat hearttype fatty acid-binding protein expressed in Escherichia coli[J].J Chromat B,1996,679:61.
[15]Dyson MR,Shadbolt SP,Vincent KJ,et al.Production of soluble mammalian proteins in Escherichia coli:identification of protein features that correlate with successful expression[J].BMC Biotechnol,2004,4:32.
[16]Braud S,Moutiez M,Belin P,et al.Dual expression system suitable for highthroughput fluorescence-based screening and production of soluble proteins[J].J Proteome Res,2005,4(6):2137-2147.
[17]Huang J,Cao L,Guo W,et al.Enhanced soluble expression of recombinant Flavobacterium heparinum heparinase I in Escherichia coli by fusing it with various soluble partners[J].Protein Express Purific,2012,83(2):169-176.
[2]Tanaka T,Hirota Y,Sohmiya K,et al.Serum and urinary human heart fatty acid-binding protein in acute myocardial infarction[J].Clin Biochem,1991,24:195.
[3]Knowlton AA,Burrier RE,Brecher P.Rabbit heart fatty acidbinding protein[J].Circul Res,1989,65:981.
[4]Gorski J,Hermens W T,Borawski J.Increased fatty acid-binding protein concentration in plasma of patients with chronic ranal failure[J].Clin Chem,1997,43:193.
[5]Keagten JA,Nieuweuhoven FAV,Dieijen Visser MPV,et al.Distribation of myoglobin and fatty acid-binding protein in human cardiac autopsises[J].Clin Chem,1996,42:337.
[6]Glatz JF,Vander Vusse GJ,Simoons ML,et al.Fatty acid-binding protein and the early detection of acute myocardial infarction[J].Clin Chim Acta,1998,272:87.
[7]Alansari SE,Croal BL.Diagnostic value of heart fatty acid binding protein and myoglobin in patients admitted with chest pain[J].Ann Clin Biochem,2004,41:391.
[8]Koichi S,Yoshihiko S,Takeshi,et al.Ongoing myocardial damage in chronic heart failure is related to activated tumor necrosis factor and fas/fas ligand system[J].Circulation J,2004,68:747.
[9]Sorensen HP,Mortensen KK.Advanced genetic strategies for recombinant protein expression in Escherichia coli[J].J Biotechnol,2005,115(2):113-128.
[10]Davis GD,Elisee C,Newham DM,et al.New fusion protein systems designed to give soluble expression in Escherichia coli[J].Biotechnol Bioeng,1999,65(4):382-388.
[11]Sorensen HP,Sperling-Petersen HU,Mortensen KK.A favorable solubility partner for the recombinant expression of streptavidin[J].Protein Express Purific,2003,32(2):252-259.
[12]Jacquet A,Daminet V,Haumont M,et al.Expression of a recombinant Toxoplasma gondii ROP2 fragment as a fusion protein in bacteria circumvents insolubility and proteolytic degradation[J].Protein Express Purific,1999,17(3):392-400.
[13]丛珊,张阳,张竹君,等.心脏型脂肪酸结合蛋白(H-FABP)原核表达、纯化及多克隆抗体制备研究[J].中国医药指南,2012,4:1-4.
[14]Schaap F,Specht B,Ger J,et al.One-step purification of rat hearttype fatty acid-binding protein expressed in Escherichia coli[J].J Chromat B,1996,679:61.
[15]Dyson MR,Shadbolt SP,Vincent KJ,et al.Production of soluble mammalian proteins in Escherichia coli:identification of protein features that correlate with successful expression[J].BMC Biotechnol,2004,4:32.
[16]Braud S,Moutiez M,Belin P,et al.Dual expression system suitable for highthroughput fluorescence-based screening and production of soluble proteins[J].J Proteome Res,2005,4(6):2137-2147.
[17]Huang J,Cao L,Guo W,et al.Enhanced soluble expression of recombinant Flavobacterium heparinum heparinase I in Escherichia coli by fusing it with various soluble partners[J].Protein Express Purific,2012,83(2):169-176.