title: Purification of TAT-CC-HA protein under native condition, and its transduction analysis and biological effects on BCR-ABL positive cells.
abstract: BCR-ABL oncoprotein is the cause of chronic myeloid leukemia. The homologous oligomerization of BCR-ABL protein mediated by BCR coiled-coil (CC) domain plays an important role in ABL kinase activation. The HIV-1 TAT peptide has been used extensively for the introduction of proteins into cells. We recombinated a TAT-CC-HA protein to interrupt the homologous oligomerization of BCR-ABL. The expression conditions for TAT-CC-HA were optimized. The TAT-CC-HA fusion protein was purified with Ni+-NTA resin. TAT-CC-HA fusion protein was added into the cultures of Ba/F3-p210, 32D-p210, K562, KU812, Ba/F3, 32D, and HL-60 cells. It was found that TAT-CC-HA could transduce into these cells. It was confirmed that TAT-CC-HA fusion protein was internalized by Ba/F3-p210, K562, and Ba/F3 cells and located in the cytoplasm observed by confocal laser scanning fluorescence microscope. The transduction of TAT-CC-HA fusion protein into K562 cells was in a dose-dependent and time-dependent manner. The result of coimmunoprecipitation assay indicated that TAT-CC-HA could interact with BCR-ABL in K562 cells. The effects of TAT-CC-HA fusion protein on cell growth and apoptosis were detected by MTT test and flow cytometry. Our findings suggested that TAT-CC-HA fusion protein could specifically inhibit the growth of BCR-ABL positive cells, and specifically induce apoptosis of BCR-ABL positive cells, while not affect the growth and apoptosis of BCR-ABL negative cells.
authors.affiliation: Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education, Chongqing Medical University, Chongqing 400016, PR China.
authors.forename: Zhenglan
authors.initials: Z
authors.lastname: Huang
authors.forename: Maosheng
authors.initials: M
authors.lastname: Ji
authors.forename: Zhi
authors.initials: Z
authors.lastname: Peng
authors.forename: Shifeng
authors.initials: S
authors.lastname: Huang
authors.forename: Qing
authors.initials: Q
authors.lastname: Xiao
authors.forename: Chunli
authors.initials: C
authors.lastname: Li
authors.forename: Jianming
authors.initials: J
authors.lastname: Zeng
authors.forename: Miao
authors.initials: M
authors.lastname: Gao
authors.forename: Wenli
authors.initials: W
authors.lastname: Feng
mesh_terms: D017209:Apoptosis / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D016044:Fusion Proteins, bcr-abl / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D014161:Transduction, Genetic / Q000379:methods; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D016044:Fusion Proteins, bcr-abl