SARS-CoV-2 S2 variants were transiently transfected in ExpiCHO-S cells (Thermo Fisher). CHO cells were maintained and transfected according to the manufacturer’s protocols. For all ExpiCHO cultures, the manufacturer’s “High Titer” protocol was used with a 7-day culture incubation to assess relative expression. Briefly, plasmid DNA and Expifectamine were mixed in Opti-PRO SFM (Gibco) according to the manufacturer’s instructions and added to the cells. On day 1, cells were fed with manufacturer-supplied feed and enhancer as specified in the manufacturer’s protocol, and cultures were moved to a shaker incubator set to 32 °C, 5% CO2 and 115 RPM. On day 7, cultures were clarified by centrifugation, followed by addition of BioLock (IBA Life Sciences), passage through a 0.22 μM sterile filter, and purification on an KTA go system (Cytiva) using a 5mL Strep-Tactin XT column equilibrated with TBS buffer (25mM Tris pH 7.6, 200mM NaCl, 0.02% NaN3), and eluted in TBS buffer supplemented with 10mM d-desthiobiotin (Sigma Aldrich). Proteins were then purified by size-exclusion-chromatography (SEC) on a Superdex 6 Increase 10/300 column (Cytiva) in the same TBS buffer.
Biolock
Manufactured by IBA Lifesciences
Sourced in Germany
BioLock is a laboratory equipment designed to provide secure and controlled access to sensitive biological samples and materials. It features an electronic locking mechanism that can be programmed to restrict access to authorized personnel only. The core function of BioLock is to ensure the safety and integrity of critical research materials by limiting unauthorized access and maintaining a secure storage environment.
Automatically generated - may contain errors
Lab products found in correlation
D desthiobiotin, by Merck Group (1 mentions)
Optipro sfm, by Thermo Fisher Scientific (1 mentions)
Expicho s cells, by Thermo Fisher Scientific (1 mentions)
Insect xpress medium, by Lonza (1 mentions)
Drosophila s2 cells, by Thermo Fisher Scientific (1 mentions)
Pcoblast, by Thermo Fisher Scientific (1 mentions)
Effectene transfection reagent, by Qiagen (1 mentions)
Superose 6 increase 10 300 gl, by Cytiva (1 mentions)
Superose 6 increase 5 150 gl, by Cytiva (1 mentions)
Streptactin xt beads, by IBA Lifesciences (1 mentions)
A8 35, by Anatrace (1 mentions)
Esf 921 insect cell culture medium, by Expression Systems (1 mentions)
Nalgene rapid flow, by Thermo Fisher Scientific (1 mentions)
Streptrap hp column, by GE Healthcare (1 mentions)
Tskgel g4000swxl, by Tosoh (1 mentions)
5 protocols using biolock
1
Recombinant SARS-CoV-2 S2 Variant Expression
2
Recombinant Protein Expression in Drosophila S2 Cells
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Drosophila S2 cells (R69007) were purchased from Thermo Fisher Scientific and grown in Insect-Xpress medium (Lonza, Basel, Switzerland) at 28 °C. For protein expression, adherent cultures were transfected with the respective pMT/BiP expression plasmids and pCoBlast (Invitrogen, Karlsruhe, Germany) in a ratio of 20:1 using Effectene Transfection Reagent (#301425, Qiagen, Hilden, Germany) according to the instructions of the manufacturer. Stable polyclonal cell lines were subsequently selected by addition of 30 µg/mL Blasticidin and expanded to suspension cultures of 300 mL and grown at 28 °C, 80 rpm. After 5 days, the cultures were topped to 700 mL and protein expression was induced with a final concentration of 2.5 µM CdCl2. Supernatants were harvested 7 days after induction by centrifugation and were subsequently concentrated to about 50 mL using a Vivaflow 200 device (5000 MWCO PES; Sartorius, Göttingen, Germany). Biotin was blocked by addition of BioLock (IBA Lifesciences, Göttingen, Germany) as recommended. The concentrated supernatant was purified using Streptactin-Superflow high capacity slurry (IBA Lifesciences) according to the manufacturer’s protocol. All protein-containing eluates were pooled, aliquoted and stored at –80 °C until further use.
3
Protein Purification Techniques for Structural Studies
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DDM, CHS, LMNG, GDN and A8-35 were purchased from Anatrace (Maumee, OH, USA). ESF 921 Insect Cell Culture Medium was obtained from Expression Systems (Davis, CA, USA). Size exclusion chromatography columns (Superose 6 Increase 10/300 GL and Superose 6 Increase 5/150 GL) were purchased from Cytiva (Marlborough, MA, USA). Strep-Tactin®XT beads and BioLock were purchased from IBA Life Sciences (Göttingen, Germany).
4
Aerobic Expression and Purification of NifEN
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For expression of ScNifENSs ScNifENHt or ScNifENGm together with SU9-NifUAv and SU9-NifSAv, S. cerevisiae was grown in 4-L fermenters (Applikon, VERTEX Technics) under aerobic conditions (0.625 L of air per minute and liter of culture) and 250 rpm stirring as previously described (15 (link)). A typical fermenter procedure (Fig. S8) generated 200–220 g of cells that were stored in liquid N2.
STAC purifications of yeast-expressed NifEN variants were performed as described above for NifEN produced in E. coli, with the following modifications. Two hundred grams of frozen yeast pellets was resuspended in 400 mL of anaerobic buffer B (100 mM Tris-HCl, 300 mM NaCl, 10% glycerol, 2 mM DTH, and pH 8.5) supplemented with 1 mM PMSF, 1 µg/mL leupeptin, 5 µg/mL DNAse I, and 1:200 (vol/vol) BioLock (IBA Lifesciences). The cells were lysed using a HPH 2000/4-DH5 high-pressure homogenizer (IKA) operating at 20,000 psi under anaerobic conditions. The soluble protein extracts were additionally filtered by passing through a 0.2 µm pore-size filter (Nalgene Rapid-Flow, Thermo Scientific) before loading into the column. Sample loading, column washing, protein elution, NifEN concentration, buffer exchange (desalting), and protein storage were identical as in the above-described procedure.
STAC purifications of yeast-expressed NifEN variants were performed as described above for NifEN produced in E. coli, with the following modifications. Two hundred grams of frozen yeast pellets was resuspended in 400 mL of anaerobic buffer B (100 mM Tris-HCl, 300 mM NaCl, 10% glycerol, 2 mM DTH, and pH 8.5) supplemented with 1 mM PMSF, 1 µg/mL leupeptin, 5 µg/mL DNAse I, and 1:200 (vol/vol) BioLock (IBA Lifesciences). The cells were lysed using a HPH 2000/4-DH5 high-pressure homogenizer (IKA) operating at 20,000 psi under anaerobic conditions. The soluble protein extracts were additionally filtered by passing through a 0.2 µm pore-size filter (Nalgene Rapid-Flow, Thermo Scientific) before loading into the column. Sample loading, column washing, protein elution, NifEN concentration, buffer exchange (desalting), and protein storage were identical as in the above-described procedure.
5
Purification of S Protein-Fab Complexes
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For electron microscopy imaging of S protein in complex with Fab forms of human mAbs, we expressed a variant of S6Pecto protein containing a C-terminal Twin-Strep-tag, similar to that described previously (Zost et al., 2020b (link)). Expressed protein was incubated with BioLock (IBA Lifesciences) and then isolated by Strep-tag affinity chromatography on StrepTrap HP columns (GE Healthcare), followed by size-exclusion chromatography on TSKgel G4000SWXL (TOSOH) if needed.
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