In vitro selection of zinc finger DNA-binding proteins through ribosome display.
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.
Ihara
Hiroshi
H
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midoriku, Yokohama 226-8501, Japan.
Mie
Masayasu
M
Funabashi
Hisakage
H
Takahashi
Fumio
F
Sawasaki
Tatsuya
T
Endo
Yaeta
Y
Kobatake
Eiry
E
D000818:Animals; D019921:Biotinylation; D045744:Cell Line, Tumor; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D051766:Early Growth Response Protein 1 / Q000737:chemistry; D015723:Gene Library; D005821:Genetic Techniques; D066298:In Vitro Techniques; D051379:Mice; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry*; D016335:Zinc Fingers
D017931:DNA Primers; D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D012333:RNA, Messenger