Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System

نویسندگان

چکیده

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with mass of approximately 350 kDa. While the cellular function FL HTT not entirely understood, mutant expansion polyQ tract above ~36 repeats associated Huntington's disease (HD), length correlating roughly age onset. To better understand effect structure on (mHTT), quantities are required. Submilligram production in mammalian cells was achieved using doxycycline-inducible stable cell line expression. However, from lines has limitations that can be overcome transient transfection methods. This paper presents robust method for low-milligram quantity and its variants codon-optimized plasmids by polyethylenimine (PEI). The scalable (>10 mg) consistently yields 1-2 mg/L culture highly purified HTT. Consistent previous reports, solution state found to dynamic; propensity form dimers high-order oligomers. A key slowing oligomer formation working quickly isolate monomeric fractions dimeric oligomeric during size exclusion chromatography. Size chromatography multiangle light scattering (SEC-MALS) used analyze dimer higher-order content No correlation observed between (Q23, Q48, Q73) content. exon1-deleted construct 91-3,144) showed comparable oligomerization 1-3,144). Production, purification, characterization methods SEC/MALS-refractive index (RI), sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot, Native PAGE, Blue PAGE described herein.

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ژورنال

عنوان ژورنال: Journal of Visualized Experiments

سال: 2021

ISSN: ['1940-087X']

DOI: https://doi.org/10.3791/63190-v