Cloning, expression, purification and functional characterization of the oligomerization domain of Bcr-Abl oncoprotein fused to the cytoplasmic transduction peptide.
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 microM. 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.
Huang
Shi-Feng
SF
Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education, Chongqing Medical University, Chongqing 400016, PR China.
Liu
Ding-Bin
DB
Zeng
Jian-Ming
JM
Xiao
Qing
Q
Luo
Miao
M
Zhang
Wen-Ping
WP
Tao
Kun
K
Wen
Jian-Ping
JP
Huang
Zong-Gan
ZG
Feng
Wen-Li
WL
D002478:Cells, Cultured; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism*; D016044:Fusion Proteins, bcr-abl / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D015870:Gene Expression; D006801:Humans; D020014:K562 Cells; D018613:Microscopy, Confocal; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*
D010455:Peptides; D011994:Recombinant Proteins; D016044:Fusion Proteins, bcr-abl