Bac to bacprotocols

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The Bac-to-Bac protocols are a set of procedures for producing recombinant baculoviruses, which are used to express foreign genes in insect cell lines. The protocols provide a streamlined workflow for generating recombinant baculoviruses and delivering the desired genes to the target insect cells.

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Lab products found in correlation

Ex cell 420 medium, by Merck Group (1 mentions) Anti flag m2 affinity agarose resin, by Merck Group (1 mentions) S 200 superdex column, by GE Healthcare (1 mentions) Hitrap q ff column, by GE Healthcare (1 mentions) Superose 6 increase column, by GE Healthcare (1 mentions) Emulsiflex c3 homogenizer, by Avestin (1 mentions)

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Bac-to-Bac

2 protocols using bac to bacprotocols

Recombinant Nectin-4 Protein Expression

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HT1376, NCI-H292, PC3, and
CT26 cells were obtained from ATCC (American Type Culture Collection).
MC38 cells were obtained from the National Cancer Institute (L-159-2018/1).
CT26 and MC38 cells were engineered to express mouse Nectin-4 (NM_027893.3)
as described.^{21 (link)} Human peripheral blood
mononuclear cell (PBMC) isolation was described.^{21 (link)}Recombinant proteins: human CD137 (92 204B, R&D
Systems) and human CD137L (2295-4L-025, R&D Systems) were purchased.
Human Nectin-4 (residues 32–349) and rat Nectin-4 (residue
31–347) with a gp67 signal sequence and C-terminal FLAG tag
were cloned into pFastbac-1 and baculovirus using standard Bac-to-Bac
protocols (Life Technologies). Sf21 cells at 1 × 10⁶ mL^–1 in Excell-420 medium (Sigma) at 27 °C
were infected at a multiplicity of infection (MOI) of 2 with a P1
virus stock for protein expression. The supernatant was harvested
at 72 h and incubated for 1 h at 4 °C with anti-FLAG M2 affinity
agarose resin (Sigma) followed by a phosphate-buffered saline (PBS)
wash. Resin was subsequently transferred to a column and washed extensively
with phosphate-buffered saline (PBS). Protein was eluted with 100
μg/mL FLAG peptide concentrated to a volume of 2 mL and loaded
onto an S-200 Superdex column (GE Healthcare) in PBS at 1 mL/min.
2 mL fractions were collected and fractions containing Nectin-4 protein
were concentrated.

Upadhyaya P., Kristensson J., Lahdenranta J., Repash E., Ma J., Kublin J., Mudd G.E., Luus L., Jeffrey P., Hurov K., McDonnell K, & Keen N. (2022). Discovery and Optimization of a Synthetic Class of Nectin-4-Targeted CD137 Agonists for Immuno-oncology. Journal of Medicinal Chemistry, 65(14), 9858-9872.

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Purification of Recombinant PRC2 Complex

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Codon-optimized versions of human Strep-EZH1, Flag-SUZ12, His-EED, and His-RBAP48 were cloned into the pBIG1a baculovirus expression plasmid, and the sequence was verified^{46 (link)}. Bacmids were generated in DH10Bac cells and used to transfect Sf9 cells using Bac-to-Bac protocols (Life Technologies). Baculovirus was used to infect Sf9 or Hi5 cells for expression and harvested 60 h post infection. Cells were lysed in Buffer A (20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10% v/v glycerol, 1 mM DTT) plus Roche cOmplete protease inhibitors using an Emulsiflex C3 homogenizer (Avestin). Lysates were clarified by centrifugation at 40,000 × g for 20 min at 4 °C, then frozen or further processed. Lysates were bound to Streptavidin beads (Strep-Tactin Macroprep, IBA) for 2 h, then washed extensively in Buffer A without protease inhibitors. Bound PRC2 was cleaved overnight with HRV3C and thrombin in Buffer A at 4 °C. Eluted protein was dialyzed into Q-low buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM DTT), bound to a HiTrap Q FF column (GE Healthcare), and eluted with a linear gradient of Q-high buffer (20 mM Tris-HCl, pH 7.5, 1 M NaCl, 1 mM DTT). Fractions containing PRC2 were pooled, concentrated, and loaded onto a Superose 6 Increase column (GE Healthcare) equilibrated with Buffer A. Fractions containing PRC2 were pooled, concentrated, and flash-frozen.

Grau D., Zhang Y., Lee C.H., Valencia-Sánchez M., Zhang J., Wang M., Holder M., Svetlov V., Tan D., Nudler E., Reinberg D., Walz T, & Armache K.J. (2021). Structures of monomeric and dimeric PRC2:EZH1 reveal flexible modules involved in chromatin compaction. Nature Communications, 12, 714.

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