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

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

Intramolecular electrostatic interactions contribute to phospholipase Cß3 autoinhibition.

Phospholipase Cß (PLCß) enzymes regulate second messenger production following the activation of G protein-coupled receptors (GPCRs). Under basal conditions, these enzymes are maintained in an autoinhibited state by multiple elements, including an insertion within the catalytic domain known as the X-Y linker. Although the PLCß X-Y linker is variable in sequence and length, its C-terminus is conserved and features an acidic stretch, followed by a short helix. This helix interacts with residues near the active site, acting as a lid to sterically prevent substrate binding. However, deletions that remove the acidic stretch of the X-Y linker increase basal activity to the same extent as deletion of the entire X-Y linker. Thus, the acidic stretch may be the linchpin in autoinhibition mediated by the X-Y linker. We used site-directed mutagenesis and biochemical assays to investigate the importance of this acidic charge in mediating PLCß3 autoinhibition. Loss of the acidic charge in the X-Y linker increases basal activity and decreases stability, consistent with loss of autoinhibition. However, introduction of compensatory electrostatic mutations on the surface of the PLCß3 catalytic domain restore activity to basal levels. Thus, intramolecular electrostatics modulate autoinhibition by the X-Y linker.