Designing repeat proteins:: Well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins
Designing repeat proteins:: Well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins
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DOI：
10.1016/s0022-2836(03)00896-9
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发表时间：
2003-09-12
影响因子：
5.6
通讯作者：
Plückthun, A
中科院分区：
文献类型：
--
作者：
Binz, HK;Stumpp, MT;Plückthun, A
我们描述了一种有效的方法来生成稳定，可溶性和表达良好的锚蛋白重复（AR）蛋白的组合文库。结合序列和结构一致性分析，我们设计了一个33个氨基酸残基AR模块，具有7个随机位置，理论多样性为7.2 X 10(7)。在N端和c端capping重复序列之间克隆不同数量的该模块，即设计用于屏蔽堆叠AR模块疏水核心的AR。通过这种方式，产生了由4到6个重复组成的设计AR蛋白组合文库，从而增强了理论上的多样性。所有随机选择的文库成员都以可溶性形式在大肠杆菌的细胞质中表达，每11瓶摇瓶培养的量高达200 mg。在一个纯化步骤中获得了几乎纯的蛋白质。所设计的AR蛋白为单体，具有与天然AR蛋白相同的CD光谱。同时，我们的AR蛋白具有高度的热稳定性，其T-m值范围从66度到85度以上。因此，我们的组合库成员具有生物技术应用所需的属性。此外，有利的生物物理性质和AR折叠的模块化可能部分解释了天然AR蛋白的丰度。(C) 2003 Elsevier Ltd.版权所有。
We describe an efficient way to generate combinatorial libraries of stable, soluble and well-expressed ankyrin repeat (AR) proteins. Using a combination of sequence and structure consensus analyses, we designed a 33 amino acid residue AR module with seven randomized positions having a theoretical diversity of 7.2 X 10(7). Different numbers of this module were cloned between N and C-terminal capping repeats, i.e. ARs designed to shield the hydrophobic core of stacked AR modules. In this manner, combinatorial libraries of designed AR proteins consisting of four to six repeats were generated, thereby potentiating the theoretical diversity. All randomly chosen library members were expressed in soluble form in the cytoplasm of Escherichia coli in amounts up to 200 mg per 11 of shake-flask culture. Virtually pure proteins were obtained in a single purification step. The designed AR proteins are monomeric and display CD spectra identical with those of natural AR proteins. At the same time, our AR proteins are highly thermostable, with T-m values ranging from 66 degreesC to well above 85 degreesC. Thus, our combinatorial library members possess the properties required for biotechnological applications. Moreover, the favorable biophysical properties and the modularity of the AR fold may account, partly, for the abundance of natural AR proteins. (C) 2003 Elsevier Ltd. All rights reserved.