Bl21 codonplus de3 ripl competent cells

The gene NMA1 was cloned from Saccharomyces cerevisiae gDNA into the protein expression vector, Pet28a (Novagen). The PetNMA1 vector was transfected into BL21-CodonPlus(DE3)-RIPL competent cells (Agilent) and protein expression induced by 0.8 mM isopropyl-β-D-thiogalactopyranoside (IPTG) when the cells reached OD₆₀₀ of 0.6~0.7 in LB media. The culture was grown for another 16 h at 37°C before the cells were pelleted and lysed with 3 freeze-thaw cycles. The protein was purified using a Ni-NTA resin (Thermo Fisher) affinity column and eluted with increasing concentrations of imidazole. The protein was aliquoted in 20% glycerol plus 2 mM DTT, flash frozen and stored at −80°C. Protein concentration was determined using Bradford assay. The protein was >95% pure as determined by SDS-PAGE gel electrophoresis.

HIV-1 GagΔp6 (MA-CA-SP1-NC, residues 1–432, plasmid HXB2) was subcloned in a pET-11a vector and expressed in BL21-CodonPlus (DE3)-RIPL competent cells (Agilent Technologies). Protein was expressed at 19 °C as described previously.^{19 (link)} Briefly, cells were grown at 37 °C in 1 L of Luria–Bertani (LB) medium at natural isotopic abundance or minimal M9 medium containing 0.3 g/L ²H/¹⁵N Isogro (Sigma-Aldrich), 99.9% (v/v) D₂O, 1 g/L ¹⁵NH₄Cl, and 3 g/L ²H₇,¹²C₆-D-glucose for ²H/¹⁵N labeling. The temperature was reduced to 19 °C, 30 min prior to incubation. Cells, induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at an optical density of A600 ≈ 0.8, were harvested 24 h after induction. Protein was purified using a combination of ion exchange and size exclusion chromatography using our published protocol.^{19 (link),34 (link)}Cloning, expression, and isolation of the mature protease (PR) and its precursor (TFR-PR) have been described before.^{35 (link),36 (link)} Induction for protein expression, isolation of inclusion bodies, and protein purification followed previously described protocols.^{37 (link),38 (link)}

The gene for NAD⁺ Synthetase, NadE, was amplified from Streptococcus pneumoniae gDNA and cloned into the Pet28a vector (Novagen) at the NdeI-BamHI sites. The PetSPNadE vector was transfected into BL21-CodonPlus(DE3)-RIPL competent cells (Agilent) and protein expression induced by 0.8 mM IPTG when the cells reached OD₆₀₀ of 0.6~0.7 in LB media. The culture was grown for another 16 h at 37^°C before the cells were pelleted and lysed with 3 freeze-thaw cycles. The protein was purified using a Ni-NTA resin affinity column and eluted with increasing concentrations of imidazole. The protein was aliquoted in 20% glycerol plus 2 mM DTT, flash frozen and stored at −80°C. Protein concentration was determined using Bradford assay. The protein was >95% pure as determined by SDS-PAGE gel electrophoresis.

Briefly, proteins were expressed in E. coli BL21 (DE3) or BL21-CodonPlus (DE3)-RIPL competent cells (Agilent). Purification of PP1 was performed as previously described (Choy et al., 2014 (link)). GADD34 was purified using heat purification (80°C, 15 min).