کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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2021252 | 1069237 | 2009 | 12 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Cloning, expression, purification and functional characterization of the oligomerization domain of Bcr–Abl oncoprotein fused to the cytoplasmic transduction peptide Cloning, expression, purification and functional characterization of the oligomerization domain of Bcr–Abl oncoprotein fused to the cytoplasmic transduction peptide](/preview/png/2021252.png)
Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require accurate cellular localization for function. Cytoplasmic transduction peptide (CTP) is a newly designed transduction peptide that carries molecules across the cell membrane with a preference to localize in the cytoplasmic compartment and is, therefore, applicable for cytoplasmic targeting. The Bcr–Abl fusion protein, playing major causative role in chronic myeloid leukemia (CML), is a cytoplasmic oncoprotein that contains an N-terminus oligomerization domain (OD) mediating homodimerization of Bcr–Abl proteins, and an intact OD in Bcr–Abl is required both for the activation of its transforming activity and tyrosine kinase. Therefore, disrupting Bcr–Abl oligomerization represents a potential therapeutic strategy for inhibiting Bcr–Abl oncogenicity. In this study, we explored the possible homodimerization-disrupting and tyrosine kinase inhibiting effect of the transduction of OD in Bcr–Abl positive K562 cells. By expressing in Escherichia coli a CTP-OD-HA fusion protein followed by Ni+–NTA affinity purification, immunoblot identification and enterokinase cleavage, we showed that the CTP-OD-HA protein was structurally and functionally active in that it potently transduced and primarily localized into the cytoplasmic compartment, heterodimerized with Bcr–Abl, and potently inhibited the phospho-tyrosine pathways of Bcr–Abl oncoprotein at a low concentration of 4 μM. These results delineate strategies for the expression and purification of therapeutic molecules for intracytoplasmic protein based therapeutics and the CTP-OD-HA-mediated killing strategy could be explored as a promising anti-leukemia agent or an adjuvant to the conventional therapeutic modalities in chronic myeloid leukemia, such as in vitro purging.
Journal: Protein Expression and Purification - Volume 64, Issue 2, April 2009, Pages 167–178