Recombinant baculoviruses were generated according to the Bac-to-Bac Baculovirus Expression System protocol (Invitrogen) with minor modifications: DH10MultiBac cells were used26 (link) and viruses were added to Sf9 cells (Invitrogen) grown in I-Max medium (Wisent Bioproducts). Proteins were expressed at 27 °C for 64 h. Subsequent steps were carried out at 4 °C. Cells were removed by centrifugation at 1,000g then at 9,000g, and supernatants were incubated with HisPur Ni-NTA resin (Thermo Fisher Scientific). The beads were washed with buffer A (50 mM Tris-HCl (pH 7.5), 500 mM NaCl and 1 mM MgCl2) and eluted with buffer A containing 250 mM imidazole-HCl. Proteins were concentrated and loaded on a Superdex 200 10/300 GL size-exclusion column (GE Healthcare) equilibrated with buffer B (15 mM Tris-HCl (pH 7.5), 100 mM NaCl and 10 μM ZnCl2). Fractions containing ASMase were applied to a Mono Q anion-exchange column (GE Healthcare) in buffer B. The flow-through was concentrated to 10 mg ml−1 and flash-frozen.
Bac to bac baculovirus expression system protocol
Manufactured by Thermo Fisher Scientific
The Bac-to-Bac Baculovirus Expression System is a protocol for producing recombinant proteins in insect cell lines. It utilizes a baculovirus transfer vector and Escherichia coli host cells to generate recombinant baculoviruses, which are then used to infect insect cells for protein expression.
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2 protocols using bac to bac baculovirus expression system protocol
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Recombinant Baculovirus Protein Expression
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Recombinant AAV Capsid Cloning and Expression
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The Cap genes of AAV1-13 serotypes were synthesized and/or cloned into pBlueScript II SK(+) plasmids between HindIII and SpeI cut sites, named as pDonor-Cap1 to pDonor-Cap13. The Cap genes of AAV1-13 serotypes are referred to AAV1 (GenBank: NC_002077.1), AAV2 (GenBank: NC_001401.2), AAV3 (GenBank: U48704.1), AAV4 (GenBank: U89790.1), AAV5 (GenBank: NC_006152.1), AAV6 (GenBank: AF028704.1), AAV7 (GenBank: AF513851.1), AAV8 (GenBank: NC_006261.1), AAV9 (GenBank: AY530579.1), AAV10 (GenBank: AY631965.1), AAV11 (GenBank: AY631966.1), AAV12 (GenBank: DQ813647.1) and AAV13 (GenBank: EU285562.1).
To construct the EF1α-GFP-WPRE-pA expression cassette, the EGFP gene was amplified with GFP-F and GFP-R primers, and then cloned into the BamHI and EcoRI cut sites of the plasmid pAAV-EF1α-Dio-GFP-WPRE-pA (addgene: 37085). The EF1α-GFP-WPRE-pA expression cassette was cut with NotI, and then inserted into pFD/Cap2-(ITR-CMV-GFP-pA)-Rep2 as previously described [36] S1. The BEVs were generated according to the Bac-to-Bac Baculovirus Expression System protocol (Invitrogen).
To construct the EF1α-GFP-WPRE-pA expression cassette, the EGFP gene was amplified with GFP-F and GFP-R primers, and then cloned into the BamHI and EcoRI cut sites of the plasmid pAAV-EF1α-Dio-GFP-WPRE-pA (addgene: 37085). The EF1α-GFP-WPRE-pA expression cassette was cut with NotI, and then inserted into pFD/Cap2-(ITR-CMV-GFP-pA)-Rep2 as previously described [36] S1. The BEVs were generated according to the Bac-to-Bac Baculovirus Expression System protocol (Invitrogen).
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