Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells
Braz. j. med. biol. res
;
45(10): 913-920, Oct. 2012. ilus
Article
in English
| LILACS
| ID: lil-647752
ABSTRACT
The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.
Full text:
Available
Index:
LILACS (Americas)
Main subject:
Gene Products, tat
/
Cytoplasm
/
Leukemia Inhibitory Factor Receptor alpha Subunit
Limits:
Animals
/
Female
/
Humans
Language:
English
Journal:
Braz. j. med. biol. res
Journal subject:
Biology
/
Medicine
Year:
2012
Type:
Article
Affiliation country:
China
Institution/Affiliation country:
No. 401 Hospital of PLA/CN
/
Second Military Medical University/CN
/
State Food and Drug Administration of China/CN
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