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Increasing the library size in cDNA display by optimizing purification procedures
Yuki Mochizuki1, Shigefumi Kumachi, Koichi Nishigaki
1Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan. nemoto@fms.saitama-u.ac.jp.
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.
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