Improvement of a puromycin-linker to extend the selection target varieties in cDNA display method
| Autor: | Naoto Nemoto, Shingo Ueno, Shinnosuke Kimura, Takanori Ichiki |
|---|---|
| Rok vydání: | 2012 |
| Předmět: |
Ribonucleotide
DNA Complementary RNase P Bioengineering General Medicine Protein engineering Biology Directed evolution Protein Engineering Applied Microbiology and Biotechnology Molecular biology Polyethylene Glycols Biochemistry Complementary DNA mRNA display Puromycin RNA Messenger Directed Molecular Evolution Nucleic acid analogue Linker Biotechnology Gene Library |
| Zdroj: | Journal of biotechnology. 162(2-3) |
| ISSN: | 1873-4863 |
| Popis: | cDNA display using a puromycin-linker to covalently bridge a protein and its coding cDNA is a stable and efficient in vitro protein selection method. The optimal design of the often-used puromycin-linker is vital for effective selection. In this report, an improved puromycin-linker containing deoxyinosine bases as cleavage sites, which are recognized by endonuclease V, was introduced to extend the variety of the selection targets to molecules such as RNA. The introduction of this linker enables efficient in vitro protein selection without contamination from RNase T1, which is used for the conventional linker containing ribonucleotide G bases. In addition, mRNA-protein fusion efficiency was found to not depend on the length of the flexible poly (ethylene glycol) (PEG) region of the linker. These findings will allow practical and easy-to-use in vitro protein selection by cDNA display. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect