Citation
- Authors: Dai DL. et al.
- Year: 2021
- Journal: Biochemistry 60 1808-1821
- Applications: in vitro / DNA / FectoPRO
- Cell type: HEK-293F
Method
A 150 mL HEK293F culture was transfected with 37.5 μg of each TSC1, 3×-FLAG-TSC2 (N1643Q), and TBC1D7 using FectoPRO transfection agent at a ratio of 1 μL/μg of DNA. The cell density was 0.8 × 106 cells/mL with 99% viability before transfection. Cells were harvested 48 post-transfection and stored at −80 °C until they were used.
A 25 mL HEK293F starter cell culture in Gibco FreeStyle medium was grown in a New Brunswick S41i shaking incubator at 37 °C, 8% CO2, and 125 rpm until it reached a total cell density of 3.0 × 106 cells/mL and a viability of at least 95%. The starter culture was then used to seed a 22.5 mL transfection culture to a total cell density of 0.8 × 106 cells/mL. The transfection culture was then grown in a New Brunswick S41i shaking incubator at 37 °C, 8% CO2, and 125 rpm for 5 h until the total cell density reached 1.11 × 106 cells/mL. The transfection culture was then mixed with a filtered 2.5 mL transfection solution in Gibco FreeStyle medium composed of 6.25 μg of anti-TSC2 Fab light chain DNA, 6.25 μg of Fab heavy chain DNA, and 12.5 μL of FectoPRO (Polyplus Transfection, reference 166-001, lot 12W2808FCI).
Abstract
Tuberous sclerosis protein complex (pTSC) nucleates a proteinaceous signaling hub that integrates information about the internal and external energy status of the cell in the regulation of growth and energy consumption. Biochemical and cryo-electron microscopy studies of recombinant pTSC have revealed its structure and stoichiometry and hinted at the possibility that the complex may form large oligomers. Here, we have partially purified endogenous pTSC from fasted mammalian brains of rat and pig by leveraging a recombinant antigen binding fragment (Fab) specific for the TSC2 subunit of pTSC. We demonstrate Fab-dependent purification of pTSC from membrane-solubilized fractions of the brain homogenates. Negative stain electron microscopy of the samples purified from pig brain demonstrates rod-shaped protein particles with a width of 10 nm, a variable length as small as 40 nm, and a high degree of conformational flexibility. Larger filaments are evident with a similar 10 nm width and a ≤1 μm length in linear and weblike organizations prepared from pig brain. Immunogold labeling experiments demonstrate linear aggregates of pTSC purified from mammalian brains. These observations suggest polymerization of endogenous pTSC into filamentous superstructures.