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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

Authors :
Ihara, H.
mie, masayasu
Funabashi, H.
Takahashi, F.
Sawasaki, T.
Endo, Y.
KOBATAKE, EIRY
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.

Details

Language :
English
ISSN :
0006291X
Volume :
345
Issue :
No. (3)
Database :
OpenAIRE
Journal :
Biochemical and Biophysical Research Communications
Accession number :
edsair.doi.dedup.....841fd3d7e87c20eed3725755f41a8e3d