Scalable Production in Human Cells and Biochemical Characterization of Full-Length Normal and Mutant Huntingtin
| Autor: | Stefan Kochanek, Johannes Buchner, Tanja Lucas, Hans Voshol, Claudia Kueppers, Bin Huang, Xiaomin Dong, Alexander Bepperling, Andreas Weiss, Bertran Gerrits, Maike Krause |
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| Rok vydání: | 2015 |
| Předmět: |
congenital
hereditary and neonatal diseases and abnormalities Huntingtin Mutant lcsh:Medicine Nerve Tissue Proteins Biology medicine.disease_cause Protein Structure Secondary Cell Line Exon Protein structure mental disorders Huntingtin Protein medicine Humans Phosphorylation lcsh:Science Gene Mutation Multidisciplinary Circular Dichroism lcsh:R Recombinant Proteins ddc nervous system diseases nervous system Biochemistry Cytoplasm Doxycycline lcsh:Q Protein Processing Post-Translational Research Article |
| Zdroj: | PLoS ONE PLoS ONE, Vol 10, Iss 3, p e0121055 (2015) |
| ISSN: | 1932-6203 |
| DOI: | 10.1371/journal.pone.0121055 |
| Popis: | Huntingtin (Htt) is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington's disease (HD) is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ) expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q) and mutant (46Q and 128Q) Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on "gutless" adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs) were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different. |
| Databáze: | OpenAIRE |
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