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Scalable production of full-length normal and mutant huntingtin in human cells and its biochemical characterization

Bin, Huang and Tanja, Lucas and Claudia, Küppers and Xiaomin, Dong and Maike, Krause and Alexander, Bepperling and Johannes, Buchner and Voshol, Johannes and Andreas, Weiss and Gerrits, Bertran and Stefan, Kochanek (2014) Scalable production of full-length normal and mutant huntingtin in human cells and its biochemical characterization. PLOS ONE.

Abstract

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.

Item Type: Article
Keywords: Huntingtin
Date Deposited: 28 Apr 2016 23:45
Last Modified: 28 Apr 2016 23:45
URI: https://oak.novartis.com/id/eprint/23946

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