Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer.
Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer.
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DOI：
10.1186/1472-6750-9-37
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发表时间：
2009-04-21
影响因子：
3.5
通讯作者：
Stewart L
中科院分区：
文献类型：
--
作者：
Raymond A;Lovell S;Lorimer D;Walchli J;Mixon M;Wallace E;Thompkins K;Archer K;Burgin A;Stewart L
为了提高异源蛋白质生产的产量和成功率，我们开发了数据库和算法软件包Gene Composer。这个免费提供的电子工具促进了蛋白质构建体及其工程合成基因序列的信息丰富的设计，如所附手稿中详细说明的那样。在这份报告中，我们比较了异源蛋白质表达水平从天然序列的密码子工程设计的基因合成的基因结构基因作曲家。一组蛋白质包括人激酶（P38α）、病毒聚合酶（HCV NS5B）和细菌结构蛋白（FtsZ）在两种E. coli和无细胞麦胚翻译系统。我们还比较了E.大肠杆菌中的一组11种不同的蛋白质，具有很大不同的G：C含量和密码子偏好。结果一致地证明，来自密码子工程化的Gene Composer设计的蛋白质产率与从同义天然基因获得的那些一样好或更好。此外，借助Gene Composer设计的结构指导的N末端和C末端缺失构建体可以在基因到结构工作中取得更大的成功，例如来自枯草杆菌的FtsZ的X射线晶体结构测定。这些结果验证了基因合成器算法，并表明，使用合成基因和蛋白质构建工程工具的组合可以提高基因结构研究的经济性。
With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38α), viral polymerase (HCV NS5B), and bacterial structural protein (FtsZ) were expressed in both E. coli and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.
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影响因子：
7
作者：
Lithwick, G;Margalit, H
通讯作者：
Margalit, H
DOI：
10.1007/s10969-007-9033-4
发表时间：
2007-12-01
期刊：
Journal of Structural and Functional Genomics
影响因子：
--
作者：
Kefala, Georgia;Kwiatkowski, Witek;Choe, Senyon
通讯作者：
Choe, Senyon
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
3.2
作者：
BOUADLOUN, F;SRICHAIYO, T;BJORK, GR
通讯作者：
BJORK, GR
DOI：
10.1007/bf00328050
发表时间：
1984-01-01
期刊：
MOLECULAR AND GENERAL GENETICS
影响因子：
--
作者：
HAGERVALL, TG;BJORK, GR
通讯作者：
BJORK, GR