Bac to bac

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The Bac-to-Bac system is a tool used for the production of recombinant baculoviruses, which can be used to express proteins in insect cell lines. The system involves the use of a bacterial plasmid that contains the gene of interest, which is then used to generate a recombinant baculovirus that can be used to infect insect cells and produce the desired protein.

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38 protocols using bac to bac

Expression and Purification of Aurora Kinases

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Example 21

Expression and Purification of Aurora Kinase Enzymes

Aurora a Enzyme Expression and Purification

Recombinant mouse Aurora A with an amino-terminus hexahistidine tag (His-Aurora A) was expressed using a standard baculovirus vector and insect cell expression system (Bac-to-Bac®, Invitrogen).

Soluble, recombinant mouse Aurora A was purified from insect cells using Ni-NTA agarose (Qiagen) as described by the manufacturer and further purified over an S75 size exclusion column (Amersham Pharmacia Biotech).

Aurora B Enzyme Expression and Purification

Recombinant mouse Aurora B with an amino-terminus hexahistidine tag (His-Aurora B) was expressed using a standard baculovirus vector and insect cell expression system (Bac-to-Bac®, Invitrogen).

Soluble, recombinant mouse Aurora B was purified from insect cells using Ni-NTA agarose (Qiagen) as described by the manufacturer.

US11014928B2. Compounds and methods for inhibiting mitotic progression (2021-05-25). Millennium Pharmaceuticals, Inc. [US]. Inventors: Christopher F. Claiborne [CH], Lloyd J. Payne [GB], Richard J. Boyce [GB], Todd B. Sells [US], Stephen G. Stroud [US], Stuart Travers [GB], Tricia J. Vos [US], Gabriel S. Weatherhead [US].

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Purification of Recombinant Aurora A and B

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Example 4

Aurora A Enzyme Expression and Purification

Recombinant mouse Aurora A with an amino-terminus hexahistidine tag (His-Aurora A) was expressed using a standard baculovirus vector and insect cell expression system (Bac-to-Bac®, Invitrogen).

Aurora B Enzyme Expression and Purification

Recombinant mouse Aurora B with an amino-terminus hexahistidine tag (His-Aurora B) was expressed using a standard baculovirus vector and insect cell expression system (Bac-to-Bac®, Invitrogen).

Soluble, recombinant mouse Aurora B was purified from insect cells using Ni-NTA agarose (Qiagen) as described by the manufacturer.

US09988384B2. Aurora kinase inhibitors for inhibiting mitotic progression (2018-06-05). None [US], None [US], None [US]. Inventors: Christopher F. Claiborne [US], Todd B. Sells [US], Stephen G. Stroud [US].

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Expression and Purification of Human TRPV3

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For expression in Sf9 insect cells (Spodoptera frugiperda), cDNA encoding human TRPV3 (hTRPV3) was subcloned into the pFastBac vector, and baculovirus was produced according to the manufacturer's protocol (Bac-to-Bac, Invitrogen). The hTRPV3 construct was expressed as a fusion protein with a PreScission cleavage site, C-terminal green fluorescent protein (GFP), and His₈ tag. Sf9 cells were harvested 60 h postinfection by centrifugation (10,000 × g for 20 min) and resuspended in buffer A (200 mm NaCl, 50 mm Tris, pH 7.5), supplemented with 10 μg·ml⁻¹ DNase, 5 mm MgCl₂, and protease inhibitors (1 mm phenylmethanesulfonyl fluoride, 1 μg·ml⁻¹ pepstatin, 1 μg·ml⁻¹ leupeptin, and 1 μg·ml⁻¹ aprotinin). The resuspended cells were subsequently lysed by sonication and centrifuged (10,000 × g for 20 min) to discard unbroken cells. Membranes were harvested by ultracentrifugation (125,000 × g for 1 h) and resuspended in buffer A, supplemented with protease inhibitors (1 ml of buffer/g of membrane). Homogenized membranes were sampled in 360-μl aliquots, snap-frozen in liquid N₂, and stored at −80 °C until further use.

Billen B., Brams M., Debaveye S., Remeeva A., Alpizar Y.A., Waelkens E., Kreir M., Brüggemann A., Talavera K., Nilius B., Voets T, & Ulens C. (2015). Different Ligands of the TRPV3 Cation Channel Cause Distinct Conformational Changes as Revealed by Intrinsic Tryptophan Fluorescence Quenching. The Journal of Biological Chemistry, 290(20), 12964-12974.

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Purification of Recombinant Proteins from E. coli and SF21 Cells

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Recombinant proteins were purified either from Escherichia coli or SF21 cells.
RanQ69L and x-importin-β were tagged with His-tag, produced in E. coli and purified using Ni-NTA agarose beads (Qiagen). RanQ69L was then loaded with 1 mM GTP in PBS 2 h at 4 °C. MBP-tagged x-MCRS1 was produced in E. coli and purified25 (link) using amylose resin (NEB) according to the manufacturer's recommendations.
Recombinant full-length Drosophila melanogaster KANSL1, KANSL3 and MOF were expressed with an N-terminal 3FLAG/HA tag using the Bac-to-Bac (Invitrogen) system in SF21 cells. Fifty millilitres of infected cells were harvested by centrifugation, washed in PBS and resuspended in high Tween20 HKMGT buffer (25 mM HEPES pH 7.6, 150 mM KCl, 12.5 mM MgCl₂, 10% glycerol, 0.4% Tween20) with Complete protease inhibitors (Roche) added. Following three freeze thaw cycles between liquid nitrogen and a 20 °C water bath, the lysate was ultracentrifuged at 100,000g for 30 min at 4 °C. The cleared lysate was incubated with 80 μl pre-washed FLAG beads (Sigma) overnight. Following one wash in the above HKMGT buffer and three in a low Tween20 (0.1%) HKMGT buffer, the samples were eluted using 250 μg ml⁻¹ of 3XFLAG peptide (Sigma) in low Tween20 HKMGT with protease inhibitors added. Proteins were quantified by Coomassie blue staining and snap frozen in aliquots.

Meunier S., Shvedunova M., Van Nguyen N., Avila L., Vernos I, & Akhtar A. (2015). An epigenetic regulator emerges as microtubule minus-end binding and stabilizing factor in mitosis. Nature Communications, 6, 7889.

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Generating CtIP Wild-Type and Mutant Proteins

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An expression construct for CtIP-wt was generated by PCR from a pBSK-CtIP construct (gift from W.-H. Lee, University of California, Irvine School of Medicine, Irvine, CA) to generate an N-terminal, Flag-tagged wt allele in pFastBac1 (Invitrogen). S327A and T847A mutants were generated by mutagenesis (QuikChange; Agilent Technologies). Transfer vectors were converted into corresponding bacmids and were used to make the virus according to the manufacturer’s instructions for the baculovirus expression system (Bac-to-Bac; Invitrogen). The wt and mutant CtIP proteins were expressed in Sf21 insect cells.

Barton O., Naumann S.C., Diemer-Biehs R., Künzel J., Steinlage M., Conrad S., Makharashvili N., Wang J., Feng L., Lopez B.S., Paull T.T., Chen J., Jeggo P.A, & Löbrich M. (2014). Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1. The Journal of Cell Biology, 206(7), 877-894.

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Receptor and G-Protein Expression in Insect Cells

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The human β₂AR receptor was expressed in Spodoptera frugiperda (Sf9) cells infected with recombinant baculovirus (BestBac, Expression Systems). Human Gs heterotrimer was expressed in Trichoplusia ni insect cells with recombinant baculovirus (Bac-to-Bac, Invitrogen). For cAMP Glosensor assay and NanoBiT-G-protein dissociation assay, HEK293 cells deficient for β₂AR and β-arrestin1/2^{42 (link)} were grown in a humidified 37 C incubator with 5% CO₂ using Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific). E. coli BL21(DE3) cells were used to express GST-CT fusion protein, mini-Gas, Nb6B9, and SrtA enzyme.

Heng J., Hu Y., Pérez-Hernández G., Inoue A., Zhao J., Ma X., Sun X., Kawakami K., Ikuta T., Ding J., Yang Y., Zhang L., Peng S., Niu X., Li H., Guixà-González R., Jin C., Hildebrand P.W., Chen C, & Kobilka B.K. (2023). Function and dynamics of the intrinsically disordered carboxyl terminus of β2 adrenergic receptor. Nature Communications, 14, 2005.

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Baculovirus Expression of γ-Tubulin

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pACEBAC1–γ-tubulin–TEV–HIS6 WT or N229A plasmids were transformed into DH10MultiBacTurbo cells, isolated, and transfected into Sf9 cells according per the Bac-to-Bac manual (Invitrogen). Baculoviruses were amplified twice, and fresh P3 virus was used to infect 2 liters of High Five cells (Thermo Fisher Scientific) at a 1:75 dilution and a cell density of 2.5 × 10⁶/ml for 60 h at 27°C. Purification of γ-tubulin was performed as described previously (Rice et al., 2008 (link)).

Berman A.Y., Wieczorek M., Aher A., Olinares P.D., Chait B.T, & Kapoor T.M. (2023). A nucleotide binding–independent role for γ-tubulin in microtubule capping and cell division. The Journal of Cell Biology, 222(3), e202204102.

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FAMK-1 Protein Purification and Antibody Generation

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FAMK-1 (residues 60–512) was expressed using a modified pI-secSUMOstar vector (LifeSensors) and purified from Hi5 insect cells (Bac-to-Bac, B85502; Invitrogen; Xiao et al., 2013 (link)). The purified protein was sent to Cocalico Biologicals for immunizing rabbits. FAMK-1–specific antibodies were affinity purified from serum with a HiTrap NHS-activated affinity column (17-0716-01; GE Healthcare) on which FAMK-1 was immobilized.

Gerson-Gurwitz A., Worby C.A., Lee K.Y., Khaliullin R., Bouffard J., Cheerambathur D., Oegema K., Cram E.J., Dixon J.E, & Desai A. (2019). Ancestral roles of the Fam20C family of secreted protein kinases revealed in C. elegans. The Journal of Cell Biology, 218(11), 3795-3811.

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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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Recombinant Expression and Purification of KIT D4D5

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Different DNA constructs of human KIT D4D5 fragments (aa 308-514 and 308-507) were used in this study: KIT D4D5 variants with noncleavable 6xHis tag at the C-terminus were used in the light scattering experiments, and D4D5 variants with N-terminus 6xHis tag followed by TEV cleavage site were used for crystallization (see Supplementary Material). All constructs contained the original signal sequence of human KIT. The cDNAs were cloned into pFastBac1 (Invitrogen) by NcoI and HindIII restriction sites. Site directed mutagenesis was used to generate oncogenic variants of KIT D4D5.
Soluble KIT D4D5 fragments were expressed in Sf9 insect cells according to the Bac-to-Bac instruction manual (Invitrogen). For light scattering experiments KIT D4D5 fragments were purified by Ni-affinity followed and size exclusion chromatographies. The D4D5 fragments of KIT used for crystallization was partially deglycosylated using recombinant Endoglycosidase F1 and additionally purified by anion exchange and size exclusion chromatography. For details see Supplemental Experimental Procedures.

Reshetnyak A.V., Opatowsky Y., Boggon T.J., Folta-Stogniew E., Tome F., Lax I, & Schlessinger J. (2014). The Strength And Cooperativity of Contacts in KIT Extracellular Domain Determine Normal Ligand Dependent Stimulation or Oncogenic Activation in Cancer. Molecular cell, 57(1), 191-201.

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