His6-TEVsite-tagged and MBP-His6-tagged Kif18A fragments were expressed in E.coli BL21-RIL, mGFP-His10-tagged Kif18A in SF9 insect cells using the Bac-to-Bac Baculovirus system (Invitrogen). Proteins were purified using Ni-IDA Resin (Macherey-Nagel). Gel filtration was performed using an Äkta-Purifier FPLC and Superdex-200 10/300 and Superose-6 10/300 as described in Möckel et al. (2016) (link).
Bac to bac baculovirus system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Bac-to-Bac baculovirus system is a tool for the expression of recombinant proteins in insect cells. It provides a method for the generation of recombinant baculoviruses and the subsequent expression of target proteins in insect cell cultures.
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Lab products found in correlation
Cellfectin 2 reagent, by Thermo Fisher Scientific (7 mentions)
Pfastbac dual vector, by Thermo Fisher Scientific (7 mentions)
Sf9 insect cells, by Thermo Fisher Scientific (3 mentions)
Superdex 200 column, by GE Healthcare (2 mentions)
Ni nta sepharose, by GE Healthcare (1 mentions)
Cholesteryl hemisuccinate, by Merck Group (1 mentions)
N dodecyl β d maltopyranoside, by Thermo Fisher Scientific (1 mentions)
Talon imac resin, by Takara Bio (1 mentions)
Edta free protease inhibitor cocktail, by Roche (1 mentions)
Pfastbac1, by Thermo Fisher Scientific (1 mentions)
Hi5 insect cells, by Thermo Fisher Scientific (1 mentions)
Sim hf medium, by Sino Biological (1 mentions)
Source 15q, by GE Healthcare (1 mentions)
Ni nta, by Qiagen (1 mentions)
Nickel nitrilotriacetic acid resin, by GE Healthcare (1 mentions)
Clonexpress 2 one step cloning kit, by Vazyme (1 mentions)
In fusion cloning, by Takara Bio (1 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Biacore evaluation software, by GE Healthcare (1 mentions)
Cm5 chip, by GE Healthcare (1 mentions)
34 protocols using bac to bac baculovirus system
1
Purification of Kif18A Fragments
2
SARS-CoV-2 RBD Protein Production
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Recombinant RBDs and the N-terminal peptidase domain of human ACE2 (residues Ser19 to Asp615) were expressed using the Bac-to-Bac Baculovirus System (Invitrogen) as previously described26 (link). Specifically, SARS-CoV-2 RBD (residues Arg319 to Lys529) containing the gp67 secretion signal peptide and a C-terminal hexahistidine was inserted into pFastBac-Dual vectors (Invitrogen) and transformed into DH10 Bac component cells (Supplementary Table 3 ). The recombinant bacmid was extracted and further transfected into Sf9 cells using Cellfectin II Reagents (Invitrogen). The recombinant viruses were harvested from the transfected supernatant and amplified to generate high-titer virus stock. Viruses were then used to infect Sf9 cells for RBD expression. Secreted RBD was harvested from the supernatant and purified by gel filtration chromatography.
3
Recombinant SARS-CoV-2 RBD and ACE2 Expression
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Recombinant wild-type RBD, RBD with 417N-484K-501Y mutations, and human receptor ACE2 peptidase domain were expressed using the Bac-to-Bac Baculovirus System (Invitrogen) as previously described (Lan et al., 2020 (link)). Specifically, SARS-CoV-2 RBD (residues Arg319 to Lys529) or ACE2 (residues Ser19 to Asp615) containing the gp67 secretion signal peptide and a C-terminal hexahistidine was inserted into pFastBac-Dual vectors (Invitrogen) and transformed into DH10 Bac component cells. The recombinant bacmid was extracted and further transfected into Sf9 cells using Cellfectin II Reagents (Invitrogen). The recombinant viruses were harvested from the transfected supernatant and amplified to generate high-titer virus stock. Viruses were then used to infect Sf9 cells for protein expression. Secreted RBD and ACE2 were harvested from the supernatant, captured by Ni-NTA Sepharose (GE Healthcare) and purified by gel filtration chromatography.
4
Purification and Crystallization of GLP-1R Constructs
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The new GLP-1R constructs (M9, M8, M6) containing the same expression cassette as M10 were purified and crystallized as previously described (Song et al., 2017 ▸ ). Briefly, the Bac-to-Bac Baculovirus System (Invitrogen) in Spodoptera frugiperda cells was used for expression. Cells were infected at a density of 2–3 × 106 cells ml−1, grown at 27°C, and harvested 48 h after infection. After two washes of low-salt buffer and three washes of high-salt buffer, the cell membrane was solubilized in the presence of 200 µM ligand (PF-06372222), 2 mg ml−1 iodoacetamide and (EDTA)-free protease inhibitor cocktail (Roche). 1.0%(w/v) DDM (n-dodecyl-β-d -maltopyranoside, Affymetrix) and 0.2%(w/v) CHS (cholesteryl hemisuccinate, Sigma-Aldrich) were used for the solubilization, and the supernatant was then collected and incubated with TALON IMAC resin (Clontech, Palo Alto, USA) at 4°C, overnight. After washing, the resin was resuspended and treated with tobacco etch virus protease (TEV), and the receptor protein was harvested in the flow through with buffer [25 mM HEPES, pH 7.5, 500 mM NaCl, 5%(v/v) glycerol, 0.01%(w/v) DDM, 0.002%(w/v) CHS, 30 mM imidazole and 100 µM PF-06372222]. The protein was concentrated to ∼30 mg ml−1 with a 100 kDa molecular weight cutoff concentrator.
5
Phosphorylation of HDACs by CaMKII
6
Expression and Purification of SARS-CoV-2 Spike and ACE2
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The SARS-CoV-2 RBD (residues Arg319–Phe541) and the N-terminal peptidase domain of human ACE2 (residues Ser19–Asp615) were expressed using the Bac-to-Bac baculovirus system (Invitrogen) as described previously[21 (link)]. ACE2-lg, a recombinant Fc fusion protein of soluble human ACE2 (residues Gln18-Ser740), was expressed in 293F cells and purified using protein A affinity chromatography as described in our previous study[36 (link)]. The cDNA of extracellular domain (ECD) (1–1208 aa) of WT or mutant spike proteins of SARS-CoV-2 were cloned into the pCAG vector with a C-terminal T4 fibritin trimerization motif followed by a 8✕his tag and two strep tag. The recombinant protein was overexpressed using the HEK293F mammalian cells 5 days after transfection. The secreted proteins were purified by streptavidin affinity resin and size-exclusion chromatography in buffer containing 25 mM Tris (pH 8.0), 150 mM NaCl.
7
Baculovirus Expression of Calmodulin-1
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The Bac-to-Bac baculovirus system (Invitrogen) was used to express CaM1 in Sf9 cells following the manufacturer’s protocol [31] (link). Briefly, the coding motif of the CaM1 gene was ligated into the expression vector (pFastBac HTA), which was then transfected into Sf9 cells. After cultivation for 3 d at 27 °C, CaM1-expressing Sf9 cells were collected and further resuspended to measure the specific activation of PDE by CaM1.
8
Recombinant Expression and Purification of Drosophila Apoptotic Proteins
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The full-length D. melanogaster Dark complementary DNA was subcloned into vector pFastBac1 (Invitrogen) with a C-terminal 10xHis tag. The recombinant Dark was expressed using the Bac-to-Bac baculovirus system (Invitrogen). Bacmid DNAs were generated in DH10Bac cells, and the resulting baculoviruses were generated and amplified in Sf9 insect cells (Invitrogen). Dark protein was overexpressed in Hi-5 insect cells (Invitrogen) grown in the SIM HF medium (Sino Biological, Inc.). Forty-eight hours after viral infection, the cells were collected and homogenized in a buffer containing 25 mM Tris (pH 8.0) and 150 mM NaCl. The cells were disrupted by 60 strokes on ice using a Dounce homogenizer. After high-speed centrifugation at 14,000 rpm for 60 min, the supernatant was harvested. The Dark protein was purified to homogeneity by nickel affinity chromatography (Ni-NTA, Qiagen) and anion exchange chromatography (Source-15Q, GE Healthcare).
The full-length Dronc (residues 1–450), Dronc-CARD (residues 1–130), and relevant mutants were subcloned into pET-21b with a C-terminal 8xHis tag. All mutants were generated using a standard PCR-based strategy and verified by plasmid sequencing. All Dronc proteins and mutants were expressed in Escherichia coli strain BL21(DE3) and purified to homogeneity as described (Yan et al. 2006 (link)).
The full-length Dronc (residues 1–450), Dronc-CARD (residues 1–130), and relevant mutants were subcloned into pET-21b with a C-terminal 8xHis tag. All mutants were generated using a standard PCR-based strategy and verified by plasmid sequencing. All Dronc proteins and mutants were expressed in Escherichia coli strain BL21(DE3) and purified to homogeneity as described (Yan et al. 2006 (link)).
9
Cloning and Expression of Truncated Rabbit GLP-1R
10
Recombinant GLP-1R Expression in Insect Cells
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The human full-length
GLP-1R was cloned into the pFastbac 1 vector with its native signal
peptide replaced by hemeagglutinin (HA) to enhance receptor expression,
and followed by a FLAG-tag at the N terminus. A tobacco etch virus
protease (TEV) site and an 8xHis tag were appended to the C terminus
of GLP-1R. A dominant-negative Gαs (DNGαs)28 (link) was also cloned into the pFastbac 1 vector.
Human Gβ1 and human Gγ2 were cloned
into the pFastBac Dual vector. GLP-1R, DNGαs, and
Gβ1Gγ2 were coexpressed at the ratio
of 2:2:1 in Spodoptera frugiperda (Sf9) insect cells
(Invitrogen) at a density of 2 × 106 cells per mL
using the Bac-to-Bac Baculovirus system (Invitrogen). Cell pellets
were collected by centrifugation after 48 h transfection and stored
at −80 °C.
GLP-1R was cloned into the pFastbac 1 vector with its native signal
peptide replaced by hemeagglutinin (HA) to enhance receptor expression,
and followed by a FLAG-tag at the N terminus. A tobacco etch virus
protease (TEV) site and an 8xHis tag were appended to the C terminus
of GLP-1R. A dominant-negative Gαs (DNGαs)28 (link) was also cloned into the pFastbac 1 vector.
Human Gβ1 and human Gγ2 were cloned
into the pFastBac Dual vector. GLP-1R, DNGαs, and
Gβ1Gγ2 were coexpressed at the ratio
of 2:2:1 in Spodoptera frugiperda (Sf9) insect cells
(Invitrogen) at a density of 2 × 106 cells per mL
using the Bac-to-Bac Baculovirus system (Invitrogen). Cell pellets
were collected by centrifugation after 48 h transfection and stored
at −80 °C.
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