Citation

  • Authors: Dai DL. et al.
  • Year: 2021
  • Journal: Biochemistry 60(23) 1808-1821
  • Applications: in vitro / DNA / FectoPRO
  • Cell type: HEK-293F

Method

Recombinant pTSC - Expression of TSC2: A 150 mL HEK-293F culture was transfected with 37.5 μg of each TSC1, 3×-FLAG-TSC2, and TBC1D7 using FectoPRO at a ratio of 1 μL/μg of DNA. The cell density was 0.8 × 10^6 cells/mL with 99% viability before transfection. Cells were harvested 48 post-transfection and stored at −80 °C. Cells were lysed via incubation with lysis buffer. The lysate was cleared, the supernatant was filtered and passed through an anti-FLAG M2-affinity column. Expression and Purification of an Anti-TSC2 Fab: Culture was then grown in a New Brunswick S41i shaking incubator at 37 °C, 8% CO2, and 125 rpm for 5 h until cell density reached 1.11 × 10^6 cells/mL. Culture was then mixed with a filtered 2.5 mL transfection solution in Gibco FreeStyle 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. The transfection culture was grown for 96 h in the same settings as before. Culture was then decanted and centrifuged at 3000g for 10 min at 4 °C. Supernatant was collected and syringe filtered. Anti-TSC2 Fab was purified from this supernatant by gravity filtration. The elution fractions were then pooled and concentrated.

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.

Pubmed