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Bac to bac protocol

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The Bac-to-Bac protocol is a method used for the production of recombinant proteins in insect cells. It involves the use of a baculovirus expression vector system to introduce the gene of interest into insect cells, which then produce the target protein.

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Lab products found in correlation

Q5 site directed mutagenesis kit, by New England Biolabs (2 mentions) E coli strain dh10bac, by Thermo Fisher Scientific (2 mentions) Superdex g200 gel filtration column, by GE Healthcare (2 mentions) Pfastbachtb vector, by Thermo Fisher Scientific (2 mentions) Nickel affinity chromatography column, by GE Healthcare (2 mentions) Nebuilder hifi dna assembly master mix, by New England Biolabs (1 mentions) Q5 polymerase, by New England Biolabs (1 mentions) Dpni, by Thermo Fisher Scientific (1 mentions) Gateway cloning, by Thermo Fisher Scientific (1 mentions) Bac to bac, by Thermo Fisher Scientific (1 mentions)

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Bac-to-Bac (38 citations) Bac-to-Bac Baculovirus Expression System protocol (2 citations)

8 protocols using bac to bac protocol

1

Expression and purification of the ORC complex

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ORC subunits Orc1 to Orc5 (Orc1: amino acid residues 533–924, Orc2: amino acid residues 266–618, Orc3: amino acid residues 47–721, Orc4: amino acid residues 1–459, Orc5: amino acid residues 1–460) were cloned into a pFastBac-derived polycistronic BioBricks vector (UC Berkeley MacroLab). A hexa-histidine (6xHis) tag was added to the N-terminus of Orc1 and a maltose binding protein (MBP) tag to the N-terminus of Orc4, both followed by a tobacco etch virus (TEV) protease cleavage site. The C-terminus of Orc6 (amino acid residues 187–257) was cloned into a separate pFastBac vector. For Orc6 binding and crosslinking experiments, Orc6 (amino acid residues 187–257) was cloned with an N-terminal 6xHis tag into a ligation-independent-cloning (LIC)-compatible pFastBac vector14 (link). Point mutations (Y225S, A236E, and A236C) were introduced by site-directed mutagenesis and verified by DNA sequencing.
Bacmids were generated in DH10Bac cells and isolated as per the Bac-to-Bac protocol (Invitrogen Life Technologies). Sf9 cells were transfected with bacmid DNA using Cellfectin II (Invritrogen Life Technologies), also according to the manufacturer’s instructions. Baculoviruses were amplified twice in Sf9 cells before infecting large-scale cultures for protein expression.
Bleichert F., Botchan M.R, & Berger J.M. (2015). Crystal Structure of the Eukaryotic Origin Recognition Complex. Nature, 519(7543), 321-326.
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2

Engineered TRPC5 Construct Production

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All plasmids were cloned using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) and mutations were made using the Q5 Site-directed mutagenesis kit (New England Biolabs). The BacMam vector was a kind gift from Professor Eric Gouaux (Vollum Institute)44 (link). Briefly, human TRPC5 was cloned as a C-terminally truncated form (Δ766–975) with an N-terminal maltose-binding protein tag followed by a PreScission protease cleavage site (as in ref. 35 (link)) using TRPC5-SYFP231 (link) as a PCR template for TRPC5, which contains the previously described V455M mutation45 (link). Point mutations to the xanthine-binding site were introduced into TRPC5-SYFP2 in pcDNA4. Bacmids and baculoviruses were produced according to the Bac-to-Bac protocol (Invitrogen).
Wright D.J., Simmons K.J., Johnson R.M., Beech D.J., Muench S.P, & Bon R.S. (2020). Human TRPC5 structures reveal interaction of a xanthine-based TRPC1/4/5 inhibitor with a conserved lipid binding site. Communications Biology, 3, 704.
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3

Expression and purification of the ORC complex

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ORC subunits Orc1 to Orc5 (Orc1: amino acid residues 533–924, Orc2: amino acid residues 266–618, Orc3: amino acid residues 47–721, Orc4: amino acid residues 1–459, Orc5: amino acid residues 1–460) were cloned into a pFastBac-derived polycistronic BioBricks vector (UC Berkeley MacroLab). A hexa-histidine (6xHis) tag was added to the N-terminus of Orc1 and a maltose binding protein (MBP) tag to the N-terminus of Orc4, both followed by a tobacco etch virus (TEV) protease cleavage site. The C-terminus of Orc6 (amino acid residues 187–257) was cloned into a separate pFastBac vector. For Orc6 binding and crosslinking experiments, Orc6 (amino acid residues 187–257) was cloned with an N-terminal 6xHis tag into a ligation-independent-cloning (LIC)-compatible pFastBac vector14 (link). Point mutations (Y225S, A236E, and A236C) were introduced by site-directed mutagenesis and verified by DNA sequencing.
Bacmids were generated in DH10Bac cells and isolated as per the Bac-to-Bac protocol (Invitrogen Life Technologies). Sf9 cells were transfected with bacmid DNA using Cellfectin II (Invritrogen Life Technologies), also according to the manufacturer’s instructions. Baculoviruses were amplified twice in Sf9 cells before infecting large-scale cultures for protein expression.
Bleichert F., Botchan M.R, & Berger J.M. (2015). Crystal Structure of the Eukaryotic Origin Recognition Complex. Nature, 519(7543), 321-326.
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4

Purification of N-terminal Truncated SUVH9

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The N-terminal truncated SUVH9 (residues 134 - 650) was cloned into a pFastBacHT B vector (Invitrogen), which fuses a hexa-histidine tag followed by a TEV cleavage site to the N-terminus of the target gene. The plasmid was transformed into E. coli strain DH10Bac (Invitrogen) to generate the bacmid. Baculovirus was generated by transfecting Sf9 cells with the bacmid following standard Bac-to-Bac protocol (Invitrogen). The harvested virus was subsequently used to infect the suspended Hi5 cell for recombinant protein expression. The recombinant protein was first purified using nickel affinity chromatography column (GE Healthcare). The hexa-histidine tag was cleaved by TEV protease. The target protein was further purified using a Q sepharose column and a Superdex G200 gel filtration column (GE Healthcare). The purified protein was concentrated to 15 mg/ml and stored at −80°C.
Johnson L.M., Du J., Hale C.J., Bischof S., Feng S., Chodavarapu R.K., Zhong X., Marson G., Pellegrini M., Segal D.J., Patel D.J, & Jacobsen S.E. (2014). SRA/SET domain-containing proteins link RNA polymerase V occupancy to DNA methylation. Nature, 507(7490), 124-128.
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5

Baculoviral Expression of ABCG2 Variants

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The pFastBac1-BCRP vector and pENTR221-hBCRP entry vector for Gateway cloning were constructed as described previously (23 (link),24 (link)). The SNPs were incorporated into the ABCG2 gene in these vectors using the Q5 site-directed mutagenesis kit (New England Biolabs Inc., Ipswich, MA, USA) except for the 1582 G > A which was produced using the Q5 polymerase (New England Biolabs Inc.) and overlapping primers. DpnI (Thermo Fisher Scientific) was used to digest the template DNA. The presence of the SNPs was verified by sequencing the whole ABCG2 gene using the sequencing service from GATC Biotech (Constance, Germany). The ABCG2 WT and variant genes were transferred from the pENTR221-hBCRP entry vector to a modified Bac-to-Bac vector using Gateway cloning (Invitrogen). Recombinant baculoviruses for both Sf9 and HEK293 expression were generated according to the Bac-to-Bac protocol (Invitrogen Life Technologies, Carslbad, CA, USA). A vector containing the gene for enhanced yellow fluorescent protein (eYFP) was used as a control in the HEK293 expression system and an empty bacmid served as a control in the Sf9 system.
Sjöstedt N., van den Heuvel J.J., Koenderink J.B, & Kidron H. (2017). Transmembrane Domain Single-Nucleotide Polymorphisms Impair Expression and Transport Activity of ABC Transporter ABCG2. Pharmaceutical Research, 34(8), 1626-1636.
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6

Purification of N-terminal Truncated SUVH9

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The N-terminal truncated SUVH9 (residues 134 - 650) was cloned into a pFastBacHT B vector (Invitrogen), which fuses a hexa-histidine tag followed by a TEV cleavage site to the N-terminus of the target gene. The plasmid was transformed into E. coli strain DH10Bac (Invitrogen) to generate the bacmid. Baculovirus was generated by transfecting Sf9 cells with the bacmid following standard Bac-to-Bac protocol (Invitrogen). The harvested virus was subsequently used to infect the suspended Hi5 cell for recombinant protein expression. The recombinant protein was first purified using nickel affinity chromatography column (GE Healthcare). The hexa-histidine tag was cleaved by TEV protease. The target protein was further purified using a Q sepharose column and a Superdex G200 gel filtration column (GE Healthcare). The purified protein was concentrated to 15 mg/ml and stored at −80°C.
Johnson L.M., Du J., Hale C.J., Bischof S., Feng S., Chodavarapu R.K., Zhong X., Marson G., Pellegrini M., Segal D.J., Patel D.J, & Jacobsen S.E. (2014). SRA/SET domain-containing proteins link RNA polymerase V occupancy to DNA methylation. Nature, 507(7490), 124-128.
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7

Recombinant Baculovirus Production

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Production
and titering of recombinant baculoviruses were carried out using a
modified version of the standard Bac-to-Bac protocol (Invitrogen).
This has been detailed previously.25 (link)
Hauenstein A.V., Rogers W.E., Shaul J.D., Huang D.B., Ghosh G, & Huxford T. (2014). Probing Kinase Activation and Substrate Specificity with an Engineered Monomeric IKK2. Biochemistry, 53(12), 2064-2073.
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8

Expression and Purification of VyPAL2 Mutants

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The expression and purification of VyPAL2 and the different mutants were carried out as previously described (Hemu et al., 2019) . Briefly after mutagenesis, the genes coding for VyPAL and its mutants were expressed in Sf9 insect cells using the Bac-to-Bac protocol (Invitrogen). Protein purification was performed in three steps with IMAC affinity purification followed by ion-exchange and size-exclusion chromatography (SEC, with 1x PBS, pH 7.4, 1 mM DTT). The protein was then concentrated and stored at 4 °C.
Following gel filtration, proenzymes were concentrated to 2 mg/ml. The optimal activation time was estimated following a time-course analysis. Zymogens were activated at 37 ˚C in 50 mM citrate, 100 mM NaCl in a 1:1 volume ratio (v/v), with addition of 0.5 mM N-Laurocryosine, 1 mM DTT, final pH 4.4. The samples were checked using SDS-PAGE to determine the optimal activation time. After scaled-up proenzyme activation, the activation mixture was subjected to an SEC purification with buffer containing 20 mM MES, pH 6.5, 0.1 M NaCl, 1 mM DTT. Fractions containing the active form protein were pooled and concentrated for further use.
Hu S., Sahili A.E., Kishore S., Wong Y.H., Hemu X., Goh B.C., Wang Z., Tam J.P., Liu C., & Lescar J. (2021). Structural basis for substrate recognition, ligation and activation by a hyperactive Asn peptide ligase from Viola yedoensis.
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