Increasing the library size in cDNA display by optimizing purification procedures

Biological Procedures Online

Yuki Mochizuki¹  Shigefumi Kumachi¹  Koichi Nishigaki¹  Naoto Nemoto¹ 

[1] Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan

关键词: Puromycin;    Protein engineering;    mRNA/cDNA display;    In vitro protein selection;    Directed evolution;   

Others  :  793070
DOI  :  10.1186/1480-9222-15-7

 received in 2013-04-17, accepted in 2013-05-19,  发布年份 2013

【 摘 要 】

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.

【 授权许可】

   
2013 Mochizuki et al.; licensee BioMed Central Ltd.

【 图 表 】

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