Stable HEK293S cell line expressing His-tagged RBD was cultured in DMEM (high glucose, Sigma) supplemented with 10% FBS (Invitrogen), 1 mM glutamine and 1x non-essential amino acids at 37 °C. Cells were transferred to roller bottles (Greiner) and cultured in DMEM supplemented with 2% FBS, 1 mM glutamine and 1x non-essential amino acids at 30 °C for 10 days for protein expression. For protein purification, the dialyzed media was passed through a 5 mL HisTrap Nickel column (GE Healthcare). The column was washed with buffer 20 mM Tris pH 7.4, 200 mM NaCl, 30 mM imidazole and RBD was eluted using buffer 20 mM Tris pH 7.4, 200 mM NaCl, 300 mM imidazole. A volume of 30 μL endoglycosidase H1 (~1 mg ml−1) was added to ~30 mg RBD and incubated at room temperature for 2 h. Then the sample was further purified with a Superdex 75 HiLoad 16/600 gel filtration column (GE Healthcare) using 10 mM HEPES pH 7.4, 150 mM NaCl. Purified RBD was concentrated using a 10-kDa ultra centrifugal filter (Amicon) to 10.6 mg ml−1 and stored at −80°C.
Roller bottles
Manufactured by Greiner
Sourced in Belgium
Roller bottles are laboratory equipment used for the cultivation of adherent cells. They provide a controlled and efficient environment for cell growth and production. The bottles are rotated on a roller apparatus, allowing for even distribution of cells and nutrients throughout the culture medium.
Automatically generated - may contain errors
Lab products found in correlation
Histrap excel column, by GE Healthcare (2 mentions)
Histrap nickel column, by GE Healthcare (1 mentions)
T4 ligase, by New England Biolabs (1 mentions)
Superdex 200 16 60 column, by GE Healthcare (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Dh5α bacteria, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Expi293f cells, by Thermo Fisher Scientific (1 mentions)
Expi293 expression medium, by Thermo Fisher Scientific (1 mentions)
Hepes buffer, by Thermo Fisher Scientific (1 mentions)
Expicho s cells, by Thermo Fisher Scientific (1 mentions)
Expicho expression medium, by Thermo Fisher Scientific (1 mentions)
Lb media, by Merck Group (1 mentions)
Steady glo luciferase assay buffer, by Promega (1 mentions)
6 protocols using roller bottles
1
Expression and Purification of RBD Protein
2
Recircularized SV40 Vector Production
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Vector batches were produced according to Vera et al.31 (link) In brief, after digestion of SV40 expression plasmid DNA with NotI to remove the bacterial backbone, vector DNA was isolated from agarose gels and subsequently re-circularized using T4 ligase (NEB). SuperVero cells growing in roller bottles (growth area: 850 cm2; Greiner Bio One) to 20%–70% confluence in OptiPRO serum-free media containing 4 mM l -glutamine (5% carbon dioxide, 37°C) were transfected with the re-circularized vector DNA. Three days posttransfection, the culture medium containing the vector particles was collected and replaced by fresh medium. In order to further increase the number of vector particles, we used the pooled vector harvests for at least two subsequent transduction rounds. In each transduction round, SuperVero cells were transduced with 400 SV40 vector genomes per cell. Three days posttransduction, the vector particle-containing culture medium was collected and replaced by fresh media. Vector harvests were clarified and concentrated by ultracentrifugation, and stored at 4°C or other temperatures as indicated.
3
Notum Enzyme Crystallization from HEK293S Cells
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A human Notum (UniProtKB ID: Q6P988) enzyme core sequence comprising
amino acids S81–T451 with a C330S mutation was cloned into
a stable cell line vector pNeo_sec.49 (link) A
stable HEK293S GNTI- cell line50 (link) was used
for protein production for crystallization. For functional assays,
glycosylated protein was expressed in HEK293T cells. HEK cells were
expanded and grown in roller bottles (Greiner). The conditioned medium
was dialyzed and passed through a 5 ml HisTrap Excel column (GE Healthcare),
followed by 20 mM imidazole PBS wash. Notum protein was eluted with
300 mM imidazole PBS and further purified by size-exclusion chromatography
(Superdex 200 16/60 column, GE Healthcare) in 10 mM Hepes, pH 7.4,
and 150 mM NaCl buffer. To remove flexible glycans to aid crystallization,
the protein expressed in HEK293S GNTI- cells was deglycosylated with
Endo F1 (endo-β-N-acetylglucosaminidase
F1) at 37 °C, 1 h.51 (link) For crystallization,
deglycosylated Notum was concentrated to 5 mg/mL and crystallized
in 96-well Swissci/MRC plates using the sitting drop vapor diffusion
method52 (link) at 21 °C. The crystallization
drops contained 200 nL of Notum protein and 100 nL of reservoir solution
of 1.5 M ammonium sulphate and 0.1 M sodium citrate, pH 4.2.
amino acids S81–T451 with a C330S mutation was cloned into
a stable cell line vector pNeo_sec.49 (link) A
stable HEK293S GNTI- cell line50 (link) was used
for protein production for crystallization. For functional assays,
glycosylated protein was expressed in HEK293T cells. HEK cells were
expanded and grown in roller bottles (Greiner). The conditioned medium
was dialyzed and passed through a 5 ml HisTrap Excel column (GE Healthcare),
followed by 20 mM imidazole PBS wash. Notum protein was eluted with
300 mM imidazole PBS and further purified by size-exclusion chromatography
(Superdex 200 16/60 column, GE Healthcare) in 10 mM Hepes, pH 7.4,
and 150 mM NaCl buffer. To remove flexible glycans to aid crystallization,
the protein expressed in HEK293S GNTI- cells was deglycosylated with
Endo F1 (endo-β-N-acetylglucosaminidase
F1) at 37 °C, 1 h.51 (link) For crystallization,
deglycosylated Notum was concentrated to 5 mg/mL and crystallized
in 96-well Swissci/MRC plates using the sitting drop vapor diffusion
method52 (link) at 21 °C. The crystallization
drops contained 200 nL of Notum protein and 100 nL of reservoir solution
of 1.5 M ammonium sulphate and 0.1 M sodium citrate, pH 4.2.
4
Protocols for Mammalian Cell Culture and Protein Production
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DH5α bacteria (Thermo Fisher Cat# 18263012) growing in LB media (Sigma cat# L3397) at 37°C were used for cloning and amplification of plasmid DNA for mammalian cell transfection. Mammalian cells HEK293S GnTI- (ATCC® CRL-3022) or HEK293T (ATCC® CRL-3216) were grown in DMEM media supplemented with 10% fetal bovine serum (FBS, GIBCO, Cat# 12676029) at 30 or 37°C with 5 or 8% CO2 respectively. For large scale production of spike ectodomain the same type of cells were grown in roller bottles (Greiner, cat# 681075) without CO2, in DMEM media with 2% FBS at 30°C. Transient expression of RBD, ACE2, CR3022 Fab and CR3022 IgG used Expi293F cells (Thermo Fisher, Cat# A14527) grown in Expi293 Expression Medium (Thermo Fisher Cat# A1435103) in suspension with 8% CO2 at 30 or 37°C and shaking at 130 rpm. Vero E6 cells (ECACC 85020206; PHE, UK) were cultured in maintenance medium (minimum essential medium (GIBCO, Cat# 21090-022) with 2 mM L-glutamine (GIBCO, Cat# A2916801), 1% non-essential amino acids (GIBCO, Cat# 11140035), 25 mM HEPES buffer (GIBCO, Cat# 15630056) and 10% heat-inactivated (56°C for 30 min) fetal bovine serum (Sigma, Cat# F4135-500ML) at 37°C for PRNTs. ExpiCHO-S cells and ExpiCHO expression medium (Thermo Fisher, Cat# A14527) were used at 37°C with 8% CO2 for the production of CR3022 IgG for these neutralization experiments.
5
Murine Monoclonal Antibody Production for RSV
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For the analysis of antibody responses, 6–8-week-old female BALB/c mice (five to six per group) or New Zealand white rabbits (two per group) were immunized with antigen dissolved in PBS (100 µL for mice, 400 µL for rabbits). Control animals were immunized with PBS (vehicle), formalin-inactivated RSV (FI-RSV), or live RSV A2 (105 pfu). Blood was collected, and the sera were analyzed by enzyme-linked immunosorbent assay (ELISA) and by RSV nAb assays (60% PRNT and MN test) for nAbs against RSV A2 strains.
The murine mAb 10D11 was made by Eurogentec (Belgium) using V-306 as an immunogen. The spleen cells from immunized mice were fused with non-secretor Sp2/0Ag14 (ATCC® CRL8287™) myeloma cells at a 5:1 ratio, in accordance with Eurogentec (Belgium) methods. Culture supernatants were screened for anti-V-306p antibody by an ELISA and later by an RSV A Tracy virus MN test. Positive hybridomas were cloned twice by limiting dilution and expanded. For large-scale mAb production, cloned hybridoma cell lines were cultured in suspension in roller bottles (Greiner, Belgium) containing 650 mL medium, and mAb was purified by protein G high-performance affinity chromatography (GE Healthcare, Belgium). Purified mAb was dialyzed against PBS, sterile-filtered, and stored at −80 °C.
The murine mAb 10D11 was made by Eurogentec (Belgium) using V-306 as an immunogen. The spleen cells from immunized mice were fused with non-secretor Sp2/0Ag14 (ATCC® CRL8287™) myeloma cells at a 5:1 ratio, in accordance with Eurogentec (Belgium) methods. Culture supernatants were screened for anti-V-306p antibody by an ELISA and later by an RSV A Tracy virus MN test. Positive hybridomas were cloned twice by limiting dilution and expanded. For large-scale mAb production, cloned hybridoma cell lines were cultured in suspension in roller bottles (Greiner, Belgium) containing 650 mL medium, and mAb was purified by protein G high-performance affinity chromatography (GE Healthcare, Belgium). Purified mAb was dialyzed against PBS, sterile-filtered, and stored at −80 °C.
6
Purification of Notum Enzyme Core
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Methods have been described in detail in reference 18. In brief, human Notum (UniProtKB ID: Q6P988) enzyme core sequence comprising amino acids S81-T451 with a C330S mutation was cloned into a stable cell line vector pNeo_sec. A stable HEK293S GNTI-cell line was obtained and used for protein production as described previously. The cells were expanded and grown in roller bottles (Greiner). The conditioned media were dialyzed and passed through a 5 ml HisTrap Excel column (GE Healthcare), followed by 20 mM imidazole PBS wash. Notum protein was eluted with 300 mM imidazole PBS. To remove flexible glycans, the protein was wavelength of 520 nm. 20 L of steady-glo luciferase assay buffer (Promega #E2520) was added to the cell plates using a CyBio, the luminescence was measured on the PHERAstar.
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