Abstract
BackgroundThe 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.FindingsWe 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.ConclusionOur 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.
Highlights
The library size is critical for selection in evolutionary molecular engineering
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
Ribosome [2] and mRNA displays [3,4] have large libraries because the amount of mRNA-peptide/protein complex with a ribosome or puromycin is proportional to the input of mRNA (~1012/ml) in a cell-free translation system
Summary
The library size is critical for selection in evolutionary molecular engineering (directed evolution). The cDNA display method was useful for in vitro peptide and protein selection, its productivity was hindered by the generation of mRNA/cDNA-protein fusion molecules; only around 0.1% of the initial mRNA was ligated to proteins with a puromycin-linker [8]. MRNA-linker conjugate Puromycin-linker Cell-free translation mRNA-protein fusion molecule Immobilization via avidin-biotin complex and buffer-exchange
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