Highly diverse protein library based on the ubiquitous (β/α)₈ enzyme fold yields well-structured proteins through in vitro folding selection.
Highly diverse protein library based on the ubiquitous (β/α)₈ enzyme fold yields well-structured proteins through in vitro folding selection.
复制标题
DOI：
10.1002/cbic.201300326
复制
发表时间：
2013-09-02
期刊：
影响因子：
3.2
通讯作者：
Seelig, Burckhard
中科院分区：
文献类型：
--
作者：
Golynskiy, Misha V.;Haugner, John C., III;Seelig, Burckhard
正确的蛋白质折叠是蛋白质稳定性和酶活性的先决条件。虽然定向进化是研究酶功能和分离新活性的有力工具，但精心设计的折叠蛋白质文库是必不可少的。体外选择方法特别能够在数万亿蛋白质变体的文库中搜索酶活性，但缺乏具有如此高多样性的良好折叠的酶的高质量文库。我们描述了一个折叠富集蛋白质文库的构建和详细表征，该文库基于在六种酶中的五种中发现的普遍存在的（β/α）8桶折叠。我们在来自海栖热袍菌的甘油磷酸二酯磷酸二酯酶（GDPD）的单体热稳定（β/α）8桶的催化面上引入了七个随机环。我们采用了基于蛋白酶消化的体外折叠选择来富集含有三到四个折叠变体的随机环的中间文库，然后将它们组合以组装最终文库（1014个DNA序列）。使用体外蛋白酶测定法和体内GFP折叠测定法分析所得文库，其含有约1012个可溶性单体蛋白变体。我们分离了6个文库成员，并证明这些蛋白质是可溶性的，单体的，并显示出（β/α）8桶折叠样的二级和三级结构。与在没有折叠选择的情况下组装的对照文库相比，折叠富集文库的质量提高了高达50倍。据我们所知，这项工作是将超高通量方法mRNA展示与折叠选择相结合的第一个例子。由此产生的（β/α）8桶文库为研究（β/α）8折叠的独特催化能力和分离新的酶提供了有价值的起点。
Proper protein folding is a prerequisite for protein stability and enzymatic activity. While directed evolution can be a powerful tool to investigate enzymatic function and to isolate novel activities, well-designed libraries of folded proteins are essential. In vitro selection methods are particularly capable of searching for enzymatic activities in libraries of trillions of protein variants, yet high-quality libraries of well-folded enzymes with such high diversity are lacking. We describe the construction and detailed characterization of a folding-enriched protein library based on the ubiquitous (β/α)8 barrel fold found in five of the six enzyme classes. We introduced seven randomized loops on the catalytic face of the monomeric, thermostable (β/α)8 barrel of glycerophosphodiester phosphodiesterase (GDPD) from Thermotoga maritima. We employed an in vitro folding selection based on protease digestion to enrich intermediate libraries containing three to four randomized loops for folded variants and then combined them to assemble the final library (1014 DNA sequences). The resulting library was analyzed using the in vitro protease assay and an in vivo GFP-folding assay and contains ~1012 soluble monomeric protein variants. We isolated six library members and demonstrated that these proteins are soluble, monomeric and show (β/α)8 barrel fold-like secondary and tertiary structure. The quality of the folding-enriched library improved up to 50-fold compared to a control library that was assembled without the folding selection. To the best of our knowledge, this work is the first example of combining the ultra-high throughput method mRNA display with a selection for folding. The resulting (β/α)8 barrel libraries provide a valuable starting point to study the unique catalytic capabilities of the (β/α)8 fold, and to isolate novel enzymes.
登录
查看更多内容
影响因子：
14.8
作者：
Chao FA;Morelli A;Haugner JC 3rd;Churchfield L;Hagmann LN;Shi L;Masterson LR;Sarangi R;Veglia G;Seelig B
通讯作者：
Seelig B
影响因子：
3.5
作者：
Matsuura, T;Plückthun, A
通讯作者：
Plückthun, A
影响因子：
2.4
作者：
Evran, Serap;Telefoncu, Azmi;Sterner, Reinhard
通讯作者：
Sterner, Reinhard
DOI：
10.1073/pnas.0405832101
发表时间：
2004-11-23
影响因子：
11.1
作者：
Hocker, B;Claren, J;Sterner, R
通讯作者：
Sterner, R
DOI：
10.1023/b:orig.0000009836.86519.eb
发表时间：
2004-02-01
期刊：
ORIGINS OF LIFE AND EVOLUTION OF THE BIOSPHERE
影响因子：
--
作者：
Matsuura, T;Plückthun, A
通讯作者：
Plückthun, A