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In vitro selection of zinc finger DNA-binding proteins through ribosome display
In vitro selection of zinc finger DNA-binding proteins through ribosome display
- Source :
- Biochemical and Biophysical Research Communications. 345(No. (3)):1149-1154
- Publication Year :
- 2006
-
Abstract
- DNA-binding proteins with sequence specificities have a variety of applications. To create novel functional DNA-binding proteins, in vivo selection methods have been developed. There are, however, crucial problems with such methods, e.g., limitation of library size and difficulty of expression of toxic proteins for the host cells. In order to overcome these problems, we developed a novel way to select DNA-binding proteins using an in vitro ribosome display technique. The three zinc finger DNA-binding protein libraries, based on a Zif268 containing randomized sequence in each finger, were prepared and transcribed to mRNA in vitro. The ternary ribosomal complexes, formed by mRNA, ribosome, and translated DNA-binding protein during translation in a rabbit reticulocyte in vitro translation system, were selected with biotinylated target DNA fragments bound to streptavidin magnetic beads. The extracted mRNAs from the selected complexes were amplified using reverse transcription PCR and then sequenced. This is the first report of the selection of DNA-binding proteins involving an in vitro ribosome display technique.
- Subjects :
- Biophysics
In Vitro Techniques
Biology
Biochemistry
Ribosome
DNA-binding protein
Mice
Cell Line, Tumor
Animals
mRNA display
Biotinylation
RNA, Messenger
Ribosome profiling
Molecular Biology
DNA Primers
Early Growth Response Protein 1
Gene Library
Zinc finger
Zinc Fingers
Translation (biology)
Cell Biology
Ribosomal RNA
Molecular biology
DNA-Binding Proteins
Genetic Techniques
Mutation
Ribosome display
Ribosomes
Plasmids
Subjects
Details
- Language :
- English
- ISSN :
- 0006291X
- Volume :
- 345
- Issue :
- No. (3)
- Database :
- OpenAIRE
- Journal :
- Biochemical and Biophysical Research Communications
- Accession number :
- edsair.doi.dedup.....841fd3d7e87c20eed3725755f41a8e3d