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Baculogold dna

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BaculoGold DNA is a high-quality reagent used for the production of recombinant proteins in insect cell expression systems. It provides a linear baculovirus DNA template for the generation of recombinant baculoviruses.

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Lab products found in correlation

Pcdna3.1 hygromycin vector, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

BaculoGold (12 citations) BD BaculoGold Linearized Baculovirus DNA (6 citations) BaculoGold viral DNA (2 citations) BaculoGold Bright DNA (1 citations)

3 protocols using baculogold dna

1

Production and Mutagenesis of EphB2 Protein

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Mouse EphB2–ECD (residues 19–543) was cloned into a modified pAcGP67 baculovirus expression vector (BD Bioscience, Becton Dickinson Franklin Lakes Campus, NJ, USA), with GP67 secretion signal and human Fc fragment as C-terminal tag. Recombinant baculovirus was generated by co-transfecting the expression plasmid along with linearized BaculoGold DNA (BD Pharmingen Inc, San Diego, CA, USA) into SF9 cells. The human EphB2 full-length protein and the structure-based mutants were cloned into a pcDNA3.1 + hygromycin vector (Invitrogen, Waltham, MA, USA) for stable expression in a HEK293 cell line. Mutations were introduced by site-directed mutagenesis (Stratagene, San Diego, CA, USA) and were sequence-verified.
Xu Y., Robev D., Saha N., Wang B., Dalva M.B., Xu K., Himanen J.P, & Nikolov D.B. (2021). The Ephb2 Receptor Uses Homotypic, Head-to-Tail Interactions within Its Ectodomain as an Autoinhibitory Control Mechanism. International Journal of Molecular Sciences, 22(19), 10473.
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2

Recombinant Src-YEEI Kinase Production

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A human c-Src cDNA clone was modified on its C-terminal tail to encode the sequence Tyr-Glu-Glu-Ile-Pro (‘YEEI’) as described elsewhere (26 (link)). In addition, the N-terminal unique domain was replaced with a hexa-histidine tag. The resulting sequence was used to produce a recombinant baculovirus in Sf9 insect cells using BaculoGold DNA and the manufacturer’s protocol (BD Pharmingen) as previously described (27 (link)). Src-YEEI was co-expressed with the Yersinia pestis YopH phosphatase to promote dephosphorylation of the activation loop tyrosine and maintain the downregulated state (28 (link),29 (link)). Sf9 cells were grown in monolayer cultures and co-infected with the Src-YEEI and YopH baculoviruses. Cells were harvested 72 h after infection, and Src-YEEI was purified as previously described (27 (link)). Purified Src-YEEI protein was stored in 20 mM Tris–HCl, pH 8.3, containing 100 mM NaCl. Kinase protein used in the Z’Lyte in vitro kinase assays also contained 3 mM DTT. The molecular weight of purified Src-YEEI was confirmed by mass spectrometry and determined to be phosphorylated on the YEEI tail but not the activation loop (26 (link)), consistent with the structure of the downregulated conformation (6 (link)).
Moroco J.A., Baumgartner M.P., Rust H.L., Choi H.G., Hur W., Gray N.S., Camacho C.J, & Smithgall T.E. (2014). A Discovery Strategy for Selective Inhibitors of c-Src in Complex with the Focal Adhesion Kinase SH3/SH2-binding Region. Chemical biology & drug design, 86(2), 144-155.
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3

Heterologous Expression of CYP2J2 Mutants

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Wild-type CYP2J2 and the three R117 mutants in a modified pAUw51-CYPOR baculovirus expression vector were cotransfected with Baculogold DNA (BD Biosciences, Pharmingen, Rungis, France) into Spodopterafrugiperda Sf21 insect cells and amplified. These amplified stocks were used to infect Sf21 insect cells for the collection of microsomal fractions as previously described [63 (link)]. Cytochrome P450 amounts were determined as previously described [64 (link)] and the enzyme concentration was measured as the P450 fraction of the mutants (A450–A490 from the Fe(II)–CO difference spectra). The difference spectra of the Fe(II)–CO complexes did not significantly change as a function of time.
Lafite P., André F., Graves J.P., Zeldin D.C., Dansette P.M, & Mansuy D. (2018). Role of Arginine 117 in Substrate Recognition by Human Cytochrome P450 2J2. International Journal of Molecular Sciences, 19(7), 2066.
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