Increasing the library size in cDNA display by optimizing purification procedures View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2013-05-22

AUTHORS

Yuki Mochizuki, Shigefumi Kumachi, Koichi Nishigaki, Naoto Nemoto

ABSTRACT

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. More... »

PAGES

7-7

Identifiers

URI

http://scigraph.springernature.com/pub.10.1186/1480-9222-15-7

DOI

http://dx.doi.org/10.1186/1480-9222-15-7

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1042331268

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/23697943


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