214 121

Stability of Zinc Finger Nuclease Protein Is Enhanced by the Proteasome Inhibitor MG132

Title
Stability of Zinc Finger Nuclease Protein Is Enhanced by the Proteasome Inhibitor MG132
Author
Ramakrishna Suresh
Keywords
PLURIPOTENT STEM-CELLS; TARGETED GENE KNOCKOUT; HOMOLOGOUS RECOMBINATION; PATHWAY; DISRUPTION; DROSOPHILA; MUTATIONS; CLEAVAGE; ENZYMES
Issue Date
2013-01
Publisher
PUBLIC LIBRARY SCIENCE, 1160 BATTERY STREET, STE 100, SAN FRANCISCO, CA 94111 USA
Citation
PLOS ONE 권: 8 호: 1 논문 번호: e54282
Abstract
Background: Zinc finger nucleases (ZFNs) are powerful tools for gene therapy and genetic engineering. The characterization of ZFN protein stability and the development of simple methods to improve ZFN function would facilitate the application of this promising technology. However, the factors that affect ZFN protein stability and function are not yet clear. Here, we determined the stability and half-life of two ZFN proteins and examined the effect of MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal-Hl), a proteasome inhibitor, on ZFN-mediated gene modifications.Methodology/Principal Findings: ZFN proteins were expressed in 293T cells after transfection of ZFN-encoding plasmids. We studied two ZFN pairs: Z-224, which targets the CCR5 gene, and K-230, which targets a region 230 kbp upstream of CCR5. Western blotting after treatment with cycloheximide showed that the half-life of these ZFN proteins was around two hours. An immunoprecipitation assay revealed that the ZFN interacts with ubiquitin molecules and undergoes polyubiquitination in vivo. Western blotting showed that the addition of MG132, a proteasomal inhibitor, increased ZFN protein levels. Finally, a surrogate reporter assay and a T7E1 assay revealed that MG132 treatment enhanced ZFN-directed gene editing.Conclusions: To our knowledge, this is the first study to investigate ZFN protein stability and to show that a small molecule can increase ZFN activity. Our protein stability study should lay the foundation for further improvement of ZFN technology; as a first step, the use of the small molecule MG132 can enhance the efficiency of ZFN-mediated gene editing.
URI
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0054282http://hdl.handle.net/20.500.11754/41482
ISSN
1932-6203
DOI
10.1371/journal.pone.0054282
Appears in Collections:
ETC[S] > 연구정보
Files in This Item:
journal.pone.0054282.PDFDownload
Export
RIS (EndNote)
XLS (Excel)
XML


qrcode

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

BROWSE