| RRC ID |
42651
|
| 著者 |
Ihara H, Mie M, Funabashi H, Takahashi F, Sawasaki T, Endo Y, Kobatake E.
|
| タイトル |
In vitro selection of zinc finger DNA-binding proteins through ribosome display.
|
| ジャーナル |
Biochem Biophys Res Commun
|
| 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.
|
| 巻・号 |
345(3)
|
| ページ |
1149-54
|
| 公開日 |
2006-7-7
|
| DOI |
10.1016/j.bbrc.2006.05.029
|
| PII |
S0006-291X(06)01020-5
|
| PMID |
16714002
|
| MeSH |
Animals
Biotinylation
Cell Line, Tumor
DNA Primers / chemistry
DNA-Binding Proteins / chemistry*
Early Growth Response Protein 1 / chemistry
Gene Library
Genetic Techniques
In Vitro Techniques
Mice
Mutation
Plasmids / metabolism
RNA, Messenger / metabolism
Ribosomes / chemistry*
Zinc Fingers
|
| IF |
2.985
|
| 引用数 |
14
|
|
WOS 分野
|
BIOPHYSICS
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
| リソース情報 |
| ヒト・動物細胞 |
P19 |
