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Bac to bac expression system instructions manual

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The Bac to Bac expression system instructions manual provides detailed guidance on the use of the Bac to Bac expression system, a tool for the production of recombinant proteins in insect cells. The manual outlines the core functionality and procedures involved in this system, without interpretation or extrapolation on its intended use.

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Pfastbac dual, by Thermo Fisher Scientific (2 mentions)

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Bac-to-Bac system (215 citations) Bac-to-Bac (38 citations) Bac-to-Bac manual (10 citations)

2 protocols using bac to bac expression system instructions manual

1

Expression and Purification of Mutant HMPV Proteins

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HMPV L and P genes (from hMPV isolate CAN97–83, Genbank: AY297749.1)19 codon-optimized for expression in Spodoptera frugiperda 9 (Sf9) cells, were chemically synthesized and cloned into modified pFastBacDual (Thermo Fischer Scientific) by Bio Basic Inc. (Singapore). HMPV L (protein sequence Genbank: AAQ67700.1) was inserted downstream of a Strep-tag-II followed by a TEV cleavage sequence. Strep-TEV-HMPV L was inserted downstream of polyhedrin promoter on pFastBacDual. HMPV P (protein sequence Genbank: AAQ67693.1) was inserted downstream of an 8× His-tag sequence, followed by a TEV cleavage sequence. 8×His-TEV-HMPV P was inserted downstream of p10 promoter on pFastBacDual. We mutated pFastBacDual_HMPV_L:P by plasmid PCR to generate pFastBacDual_HMPV_LD745A:P using forward primer 5’-GACGTGTCCAAGCCTGTGAAGCTGTC and reverse primer 5’-GATGGACTGGTTCGCGCCGTTGAGC. The mutation was confirmed by sequencing. Generation and isolation of bacmid of HMPV_L:P and HMPV_LD745A:P followed bac to bac expression system instructions manual (Invitrogen). Viral stocks generated from purified bacmids were amplified and used for protein expression.
Pan J., Qian X., Lattmann S., Sahili A.E., Yeo T.H., Jia H., Cressey T., Ludeke B., Noton S., Kalocsay M., Fearns R, & Lescar J. (2019). Structure of the human metapneumovirus polymerase phosphoprotein complex. Nature, 577(7789), 275-279.
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2

Expression and Purification of Mutant HMPV Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
HMPV L and P genes (from hMPV isolate CAN97–83, Genbank: AY297749.1)19 codon-optimized for expression in Spodoptera frugiperda 9 (Sf9) cells, were chemically synthesized and cloned into modified pFastBacDual (Thermo Fischer Scientific) by Bio Basic Inc. (Singapore). HMPV L (protein sequence Genbank: AAQ67700.1) was inserted downstream of a Strep-tag-II followed by a TEV cleavage sequence. Strep-TEV-HMPV L was inserted downstream of polyhedrin promoter on pFastBacDual. HMPV P (protein sequence Genbank: AAQ67693.1) was inserted downstream of an 8× His-tag sequence, followed by a TEV cleavage sequence. 8×His-TEV-HMPV P was inserted downstream of p10 promoter on pFastBacDual. We mutated pFastBacDual_HMPV_L:P by plasmid PCR to generate pFastBacDual_HMPV_LD745A:P using forward primer 5’-GACGTGTCCAAGCCTGTGAAGCTGTC and reverse primer 5’-GATGGACTGGTTCGCGCCGTTGAGC. The mutation was confirmed by sequencing. Generation and isolation of bacmid of HMPV_L:P and HMPV_LD745A:P followed bac to bac expression system instructions manual (Invitrogen). Viral stocks generated from purified bacmids were amplified and used for protein expression.
Pan J., Qian X., Lattmann S., Sahili A.E., Yeo T.H., Jia H., Cressey T., Ludeke B., Noton S., Kalocsay M., Fearns R, & Lescar J. (2019). Structure of the human metapneumovirus polymerase phosphoprotein complex. Nature, 577(7789), 275-279.
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