Stability of zinc finger nuclease protein is enhanced by the proteasome inhibitor MG132.
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.


RESULTS
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.
Graduate School of Biomedical Science and Engineering/College of Medicine, Hanyang University, Seoul, South Korea.
Suresh
S
Ramakrishna
Young-Hoon
YH
Kim
Hyongbum
H
Kim
D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D057809:HEK293 Cells; D006207:Half-Life; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors / Q000494:pharmacology*; D055550:Protein Stability / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects; D019713:Receptors, CCR5 / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D025801:Ubiquitin / Q000235:genetics* / Q000378:metabolism; D054875:Ubiquitination; D016335:Zinc Fingers
D017931:DNA Primers; D007976:Leupeptins; D061988:Proteasome Inhibitors; D019713:Receptors, CCR5; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde