Tni insect cells

The truncated C-terminal ectodomain fragment of mouse TLR9 (mTLR9-cECD) was expressed in a baculovirus-insect cell system and purified as described previously (Li et al, 2012 (link)). Briefly, a gene encoding the proteolytically processed fragment (residues 474–824) mTLR9-cECD with an N-terminal eight-histidine purification tag, followed by the linker sequence Ser-Ser-Gly and a tobacco etch virus protease cleavage site, was cloned into the pAcGP67-A vector (BD Biosciences) in frame with the baculovirus gp67 signal sequence. mTLR9-cECD was expressed in Tni insect cells (Expression Systems). Cells were infected with 1% (v/v) of third-passage baculovirus stock. After culture in suspension for 72 h at 27°C mTLR9-cECD was extracted from intracellular compartments with 50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol, 1% Fos-Choline 12. mTLR9-cECD was purified by nickel affinity chromatography with a HisTrap HP column (GE Healthcare), followed by cation exchange chromatography with a MonoS column (GE Healthcare) and size-exclusion chromatography with a Superdex 200 10/300 GL column (GE Healthcare). The size-exclusion buffer was 20 mM MES pH 5.6, 100 mM NaCl, 2 mM β-mercaptoethanol.

The SecR, DNGα_sv1 or DNGαsv2 (the latter containing an additional A366S mutation)¹⁰ , Gβ₁, and Gγ₂ were expressed in Tni insect cells (Expression Systems) using baculovirus. For the first preparation, cell cultures were grown in ESF 921 serum-free media (Expression Systems) to a density of 4 million cells/mL, and then infected with three separate baculoviruses at a ratio of 4:2:1 for SecR, DNGα_sv1, and Gβ₁γ₂, respectively. In the second preparation, cell cultures were grown to a density of 3.3 million cells/mL, and then infected with the baculoviruses at a ratio of 3:2:1 for SecR, DNGα_sv2, and Gβ₁γ₂, respectively. Culture was harvested by centrifugation ~48 h post infection and cell pellet was stored at −80 °C.

Drosophila IntS4, IntS9, and IntS11 were co-expressed in insect cells. IntS9 and IntS11 were cloned into the pFL acceptor vector. N-terminal 6xHis-tagged IntS4 was cloned into the pSPL donor vector. These two vectors were fused by Cre recombinase. Tni insect cells (Expression Systems) (2 × 10⁶ cells·ml^–1) were infected with 16 ml of IntS4-IntS9-IntS11 P2 virus and harvested after 48 h.
For purification, the cell pellet was resuspended and lysed by sonication in 100 ml of buffer containing 20 mM Tris (pH 8.0), 250 mM NaCl, 2 mM βME, 5% (v/v) glycerol, and one tablet of protease inhibitor mixture (Sigma). The cell lysate was then centrifuged at 15,000 × g for 40 min at 4 °C. The protein complex was purified from the supernatant via nickel affinity chromatography (Qiagen). The protein complex was further purified using a Hiload 16/60 Superdex 200 column (Cytiva). The IntS4-IntS9-IntS11 complex was concentrated to 2 mg·ml^–1 in a buffer containing 20 mM Tris (pH 8.0), 300 mM NaCl, and 2 mM DTT, and stored at −80 °C.