通过优化纯化程序增加 cDNA 展示库的大小。
Increasing the library size in cDNA display by optimizing purification procedures.
机构信息
Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.
出版信息
Biol Proced Online. 2013 May 22;15(1):7. doi: 10.1186/1480-9222-15-7.
BACKGROUND
The library size is critical for selection in evolutionary molecular engineering (directed evolution). Although cDNA display has become a promising in vitro display technology by overcoming the instability of mRNA display, it is hindered by low yields. In this study, we improved the yield of cDNA display molecules by carefully examining each step of the preparation process.
FINDINGS
We found that steric hindrance of ribosomes binding to the mRNA-protein fusion molecules was interfering with biotin-streptavidin binding. Additionally, reducing buffer exchange by performing RNase digestion in the His-tag-binding buffer to release the cDNA display molecules improved their His-tag purification.
CONCLUSION
Our optimized conditions have improved the yield of cDNA display molecules by more than 10 times over currently used methods, making cDNA display more practically available in evolutionary molecular engineering.
背景
文库大小对于进化分子工程（定向进化）中的选择至关重要。尽管 cDNA 展示通过克服 mRNA 展示的不稳定性成为一种很有前途的体外展示技术，但它受到产量低的限制。在这项研究中，我们通过仔细检查制备过程的每一个步骤来提高 cDNA 展示分子的产量。
结果
我们发现核糖体与 mRNA-蛋白融合分子结合的空间位阻干扰了生物素-链霉亲和素结合。此外，通过在 His 标签结合缓冲液中进行 RNase 消化来减少缓冲液交换，以释放 cDNA 展示分子，从而改善了它们的 His 标签纯化。
结论
我们的优化条件使 cDNA 展示分子的产量比目前使用的方法提高了 10 多倍，使 cDNA 展示在进化分子工程中更具实际应用价值。
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