用于构建用于原子力显微镜的多聚体蛋白的通用克隆系统。
Versatile cloning system for construction of multimeric proteins for use in atomic force microscopy.
作者信息
Steward Annette, Toca-Herrera José Luis, Clarke Jane
机构信息
Department of Chemistry, MRC Centre for Protein Engineering, Lensfield Road, Cambridge CB2 1EW, UK.
出版信息
Protein Sci. 2002 Sep;11(9):2179-83. doi: 10.1110/ps.0212702.
Abstract
This manuscript introduces a versatile system for construction of multimeric proteins to be used as substrates for atomic force microscopy. The construction makes use of a cassette system that allows modules to be cut and ligated in any combination in eight different positions. The modules can be sequenced in situ after construction. A three-module fragment can be produced that is of a size amenable to structural and biophysical analysis to check the effect of placing a protein into a multimeric construct. We show that if the parent titin modules are retained in a construct, they can act both as linkers and as an internal standard for the force measurements. Proteins that cannot be expressed solubly in an eight-module homopolymer have been expressed and subject to force measurements using this system.
摘要
本手稿介绍了一种用于构建多聚体蛋白的通用系统，该系统可作为原子力显微镜的底物。构建过程利用了一种盒式系统，该系统允许模块在八个不同位置以任何组合进行切割和连接。构建完成后，模块可进行原位测序。可以产生一个三模块片段，其大小适合进行结构和生物物理分析，以检查将一种蛋白质放入多聚体构建体中的效果。我们表明，如果亲本肌联蛋白模块保留在构建体中，它们既可以作为连接体，也可以作为力测量的内部标准。使用该系统已表达了在八模块同聚物中不能以可溶形式表达的蛋白质，并对其进行了力测量。
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本文引用的文献
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Can non-mechanical proteins withstand force? Stretching barnase by atomic force microscopy and molecular dynamics simulation.非机械蛋白能承受力吗？通过原子力显微镜和分子动力学模拟拉伸核糖核酸酶抑制剂。
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