Production and Transduction of a Human Recombinant β-Globin Chain into Proerythroid K-562 Cells To Replace Missing Endogenous β-Globin.
Title | Production and Transduction of a Human Recombinant β-Globin Chain into Proerythroid K-562 Cells To Replace Missing Endogenous β-Globin. |
Publication Type | Journal Article |
Year of Publication | 2018 |
Authors | Papadopoulou, L. C., Ingendoh-Tsakmakidis A., Mpoutoureli C. N., Tzikalou L. D., Spyridou E. D., Gavriilidis G. I., Kaiafas G. C., Ntaska A. T., Vlachaki E., Panayotou G., Samiotaki M., & Tsiftsoglou A. S. |
Journal | Mol Pharm |
Volume | 15 |
Issue | 12 |
Pagination | 5665-5677 |
Date Published | 2018 12 03 |
ISSN | 1543-8392 |
Keywords | alpha-Globins, beta-Globins, beta-Thalassemia, Biological Therapy, Cell Line, Humans, Recombinant Fusion Proteins, tat Gene Products, Human Immunodeficiency Virus, Transduction, Genetic |
Abstract | Protein replacement therapy (PRT) has been applied to treat severe monogenetic/metabolic disorders characterized by a protein deficiency. In disorders where an intracellular protein is missing, PRT is not easily feasible due to the inability of proteins to cross the cell membrane. Instead, gene therapy has been applied, although still with limited success. β-Thalassemias are severe congenital hemoglobinopathies, characterized by deficiency or reduced production of the adult β-globin chain. The resulting imbalance of α-/β-globin chains of adult hemoglobin (αβ) leads to precipitation of unpaired α-globin chains and, eventually, to defective erythropoiesis. Since protein transduction domain (PTD) technology has emerged as a promising therapeutic approach, we produced a human recombinant β-globin chain in fusion with the TAT peptide and successfully transduced it into human proerythroid K-562 cells, deficient in mature β-globin chain. Notably, the produced human recombinant β-globin chain without the TAT peptide, used as internal negative control, failed to be transduced into K-562 cells under similar conditions. In silico studies complemented by SDS-PAGE, Western blotting, co-immunoprecipitation and LC-MS/MS analysis indicated that the transduced recombinant fusion TAT-β-globin protein interacts with the endogenous native α-like globins to form hemoglobin αβ-like tetramers to a limited extent. Our findings provide evidence that recombinant TAT-β-globin is transmissible into proerythroid K-562 cells and can be potentially considered as an alternative protein therapeutic approach for β-thalassemias. |
DOI | 10.1021/acs.molpharmaceut.8b00857 |
Alternate Journal | Mol Pharm |
PubMed ID | 30375878 |