膜蛋白在生物体的生命活动中发挥了非常重要的作用,如细胞的增殖和分化、能量转换、信号转导及物质运输等。据估计有大约60%的药物作用靶点是膜蛋白。然而,传统的大肠杆菌蛋白表达系统表达膜蛋白却很困难,表达产物不溶、无法开展后续实验,是影响研究进程的主要因素[1]。
延伸
CFS麦胚无细胞蛋白表达技术
表达成功率高达95%,总蛋白产量高达4mg/6mL,绝大多数表达出的蛋白具有天然的结构和活性。
无细胞蛋白表达系统通过在其反应体系中额外添加微粒体(细胞破碎后从内质网获得的人工囊泡)或脂质体(由脂质双层形成的人工囊泡),来模拟细胞内表达膜蛋白的过程[3-5]。在真核无细胞蛋白表达系统中,与兔网织红细胞或昆虫和人类细胞系的提取物相比,麦胚提取物的蛋白质产量最高。麦胚系统已成功表达多种膜蛋白[6-10],包括制备用于靶向蛋白质组学的同位素标记跨膜蛋白[11],制备针对不同GPCR的单克隆抗体[12,13]。
CFS麦胚无细胞蛋白表达系统
助您轻松表达中/大量膜蛋白
针对性强:含有脂质体,特别针对传统方法难表达、难组装的膜蛋白
操作简单:无需准备试剂, 只需几个移液步骤即可合成蛋白质
表达高效:无需培养细胞,一天内完成蛋白表达
使用方便:预分装反应试剂
01
中量无细胞膜蛋白表达试剂盒
ProteoLiposome PLUS Expression Kit
(目录号:CFS-EDX-PLUS-PLE)
试剂盒含有特殊的脂质体,通过双层液相(Bilayer)的反应形式,使目的蛋白能够在脂质体的生物膜上正确合成和组装。整个表达过程仅需4步:质粒构建--体外转录--蛋白表达--纯化,两天内即可获得目的蛋白。
操作流程
1
模板DNA构建
2
转录反应
3
翻译反应
4
样品纯化
5
验证蛋白表达
02
大量无细胞膜蛋白表达试剂盒
ProteoLiposome BD Expression Kit
试剂盒创新性地将麦胚表达系统同大豆磷脂-脂质体体系相结合,通过“双层液相+透析”的反应形式,实现膜蛋白在体外环境下的长时间(Tmax ≤72 h)高效表达[14]。
操作流程
1
模板DNA构建
2
转录反应
3
翻译反应
4
样品纯化
5
验证蛋白表达
无细胞膜蛋白大量表达实验结果
使用ProfeoLiposore BD Expression kit 表达25个膜蛋白,使用考马斯亮蓝 (CBB) 染色检测 (PAGE图)蛋白表达效果。结果显示,25个膜蛋白都能成功表达,而且表达量均在mg级别,最大可达8.5mg[15] 。
"克隆-蛋白表达" 高效解决方案
TIANGEN基因快速克隆及蛋白表达方案,让您从基因到蛋白表达的每一步,都有更快更好的选择!
CFS无细胞蛋白表达系列产品选购指南
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参考文献(滑动查看完整内容)
[1] Zhang, D., Q. Zhao, and B. Wu, Structural Studies of G Protein-Coupled Receptors. Molecules and cells, 2015. 38(10): p. 836-42.
[2] Khambhati K , Bhattacharjee G , Gohil N ,et al.Exploring the Potential of Cell-Free Protein Synthesis for Extending the Abilities of Biological Systems[J].Frontiers in Bioengineering and Biotechnology, 2019, 7:248.DOI:10.3389/fbioe.2019.00248.
[3] Banerjee, R.K. and A.G. Datta, Proteoliposome as the model for the study of membrane-bound enzymes and transport proteins. Mol Cell Biochem, 1983. 50(1): p. 3-15.
[4] Chang, C.N., G. Blobel, and P. Model, Detection of prokaryotic signal peptidase in an Escherichia coli membrane fraction: endoproteolytic cleavage of nascent f1 pre-coat protein. Proceedings of the National Academy of Sciences of the United States of America, 1978. 75(1): p. 361-5.
[5] Chang, C.N., P. Model, and G. Blobel, Membrane biogenesis: cotranslational integration of the bacteriophage f1 coat protein into an Escherichia coli membrane fraction. Proceedings of the National Academy of Sciences of the United States of America, 1979. 76(3): p.
[6] Arimitsu, E., et al., The ligand binding ability of dopamine D1 receptors synthesized using a wheat germ cell-free protein synthesis system with liposomes. European journal of pharmacology, 2014. 745: p. 117-22.
[7] Fogeron, M.L., et al., Wheat Germ Cell-Free Overexpression for the Production of Membrane Proteins. Methods Mol Biol, 2017. 1635: p. 91-108.
[8] Nozawa, A., et al., A cell-free translation and proteoliposome reconstitution system for functional analysis of plant solute transporters. Plant & cell physiology, 2007. 48(12): p. 1815-20.
[9] Nozawa, A. and Y. Tozawa, Incorporation of adenine nucleotide transporter, Ant1p, into proteoliposomes facilitates ATP translocation and activation of encapsulated luciferase. Journal of bioscience and bioengineering, 2014. 118(2): p. 130-3.
[10] Periasamy, A., et al., Cell-free protein synthesis of membrane (1,3)-beta-d-glucan (curdlan) synthase: co-translational insertion in liposomes and reconstitution in nanodiscs. Biochimica et biophysica acta, 2013. 1828(2): p. 743-57.
[11] Takemori, N., et al., High-throughput synthesis of stable isotope-labeled transmembrane proteins for targeted transmembrane proteomics using a wheat germ cell-free protein synthesis system. Molecular bioSystems, 2015. 11(2): p. 361-5.
[12] Hashimoto, Y., et al., Engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5. Sci Rep, 2018. 8(1): p. 8383.
[13] Takeda, H., et al., Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay. Scientific reports, 2015. 5: p. 11333.
[14] Takeda H , Ogasawara T , Ozawa T ,et al.Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay[J].Scientific Reports, 2015, 5:11333.DOI:10.1038/srep11333.
[15] Takeda H , Ogasawara T , Ozawa T ,et al.Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay[J].Scientific Reports, 2015, 5:11333.DOI:10.1038/srep11333.