Improvement of a puromycin-linker to extend the selection target varieties in cDNA display method

Ueno, S.; Kimura, Shinnosuke; Ichikı, Takanori; Nemoto, Naoto

doi:10.1016/j.jbiotec.2012.09.003

Cited by 10 publications

(16 citation statements)

References 11 publications

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“…Finally we achieved 17% of final yield of cDNA display molecule based on the input mRNA-linker conjugates (Figure 4B), which is more than 10 times higher than in our previous study [8,12]. Additionally, we recently also succeeded in releasing cDNA display molecules from SA-beads by using Endonuclease V instead of RNase T1 by designing a new puromycin-linker [14]. Thus, we believe that this new linker and our currently optimized conditions will make cDNA display more useful and practical for in vitro protein selection.…”

Section: Resultsmentioning

confidence: 82%

Increasing the library size in cDNA display by optimizing purification procedures

et al. 2013

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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.

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Section: Resultsmentioning

confidence: 82%

Increasing the library size in cDNA display by optimizing purification procedures

et al. 2013

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“…The DNA library was transcribed into mRNA using the T7 RiboMAX Express large scale RNA production system (Promega, Madison, WI, USA), and the synthesized mRNA was purified with an RNA purification kit (FavorPrep After Tri-18 reagent RNA clean-up kit, Favorgen, Ping-Tung, Taiwan). Purified mRNA was hybridized to a short biotin segment puromycin linker (SBP linker) 13 under annealing conditions (heating at 90 °C for 1 min followed by incubation at 70 °C for 1 min and subsequent cooling to 25 °C) in T4 ligase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 10 mM dithiothreitol, and 1 mM ATP) and ligated by T4 RNA ligase (0.4–2.0 U/pmol mRNA, Takara bio, Otsu, Japan) and polynucleotide kinase (0.5 U/pmol, Toyobo, Osaka, Japan) at 25 °C overnight. Translationof the linker-conjugated mRNAs was performed by an in vitro translation system with a Retic Lysate IVT kit (Retic Lysate IVT kit, Ambion, Austin, TX, USA) at 30 °C for 30 min.…”

Section: Methodsmentioning

confidence: 99%

An RNA Binding Peptide Consisting of Four Types of Amino Acid by in Vitro Selection Using cDNA Display

2016

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RNA–protein interactions have a central role in the living world. In this article, we examined whether primitive peptides (30 residues) consisting of four types of amino acid (Gly, Ala, Asp, and Val) could interact with tRNA as a model of primitive RNAs in the RNA world. By in vitro selection of binding peptides using the cDNA display method, a characteristic peptide was selected from a random peptide library and assayed by electrophoretic mobility shift and pull-down assays. Interestingly, the selected peptide bound to a single-stranded region including a loop structure of an RNA molecule with some sequence specificity.

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“…This technology has been successfully applied to the screening of Growth Hormone Secretagogue Receptor-binding peptide ( 49 ). Since its introduction, several studies have improved the method by making it more robust, practical and convenient through the design of new puromycin-linkers ( 50 , 51 ), a pull-down method that uses biotinylated bait protein ( 52 ) and a puromycin-linker containing 3-cyanovinylcarbazole nucleoside (cnvK) ( 53 ).…”

Section: Screening Of Biomolecules In a Single Pot Without Physical Cmentioning

confidence: 99%

High-throughput screening of biomolecules using cell-free gene expression systems

Contreras-Llano

Tan

2018

Synthetic Biology

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The incorporation of cell-free transcription and translation systems into high-throughput screening applications enables the in situ and on-demand expression of peptides and proteins. Coupled with modern microfluidic technology, the cell-free methods allow the screening, directed evolution and selection of desired biomolecules in minimal volumes within a short timescale. Cell-free high-throughput screening applications are classified broadly into in vitro display and on-chip technologies. In this review, we outline the development of cell-free high-throughput screening methods. We further discuss operating principles and representative applications of each screening method. The cell-free high-throughput screening methods may be advanced by the future development of new cell-free systems, miniaturization approaches, and automation technologies.

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Improvement of a puromycin-linker to extend the selection target varieties in cDNA display method

Cited by 10 publications

References 11 publications

Increasing the library size in cDNA display by optimizing purification procedures

Increasing the library size in cDNA display by optimizing purification procedures

An RNA Binding Peptide Consisting of Four Types of Amino Acid by in Vitro Selection Using cDNA Display

High-throughput screening of biomolecules using cell-free gene expression systems

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