Construction and maintenance of randomized retroviral expression libraries for transmembrane protein engineering.
Construction and maintenance of randomized retroviral expression libraries for transmembrane protein engineering.
复制标题
用于跨膜蛋白工程的随机逆转录病毒表达库的构建和维护。
DOI：
10.1093/protein/gzq112
复制
发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Dimaio,Daniel
中科院分区：
文献类型：
--
作者：
Marlatt,SaraA;Kong,Yong;Cammett,TobinJ;Korbel,Gregory;Noonan,JamesP;Dimaio,Daniel
从表达具有随机氨基酸片段的蛋白质的文库中进行遗传选择是鉴定具有新颖生物活性的蛋白质的有效方法。在这里，我们评估了深度 DNA 测序在表征此类随机文库的组成、多样性、大小和稳定性方面的效用。我们使用 454 焦磷酸测序对表达具有随机跨膜结构域的小蛋白的逆转录病毒文库进行测序。尽管在其构建过程中可能会发生意外的随机诱变，但测序文库编码的蛋白质的整体疏水性组成和多样性很好地符合其设计。此外，我们的测序结果使我们能够更准确地估计文库编码的不同蛋白质的数量，并表明估计随机文库大小的传统方法可能会高估其真实大小。我们的结果进一步证明，在哺乳动物细胞中表达复杂逆转录病毒文库并从这些细胞中恢复文库序列的方法中不存在显着的遗传瓶颈。这些发现表明，深度测序可用于确定其他具有随机片段的文库的质量和内容，并在选择过程中跟踪单个序列。
Genetic selection from libraries expressing proteins with randomized amino acid segments is a powerful approach to identify proteins with novel biological activities. Here, we assessed the utility of deep DNA sequencing to characterize the composition, diversity, size and stability of such randomized libraries. We used 454 pyrosequencing to sequence a retroviral library expressing small proteins with randomized transmembrane domains. Despite the potential for unintended random mutagenesis during its construction, the overall hydrophobic composition and diversity of the proteins encoded by the sequenced library conformed well to its design. In addition, our sequencing results allowed us to calculate a more accurate estimate of the number of different proteins encoded by the library and suggested that the traditional methods for estimating the size of randomized libraries may overestimate their true size. Our results further demonstrated that no significant genetic bottlenecks exist in the methods used to express complex retrovirus libraries in mammalian cells and recover library sequences from these cells. These findings suggest that deep sequencing can be used to determine the quality and content of other libraries with randomized segments and to follow individual sequences during selection.