Synthetic, insect optimized DCL2 (Genebank: CR932227 ) and DCL5 (Genebank: XM_001455443.1 ) genes (Genscript) were inserted into SacI and XbaI sites of the expression vectors pFastBac-HT (Invitrogen) or pFastBacM30 (EMBL protein facility, Heidelberg) containing either a N-terminal His-tag or GST-tag. DCL3 (Genebank: CR932226 ) and DCL4 (Genebank: CR932225 ) genes were tagged with 3xFlag-HA at the N-terminal end and cloned by PCR and ligation into pGem-T Easy Vector (Promega) containing the 5′ and 3′ regulatory sequences of PTIWI09 (Genebank: CR932246 ) to increase the expression level.
Pfastbac ht
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PFastBac™HT is a plasmid vector designed for the high-level expression of recombinant proteins in insect cells using the Baculovirus Expression Vector System (BEVS). The vector allows for the insertion of a gene of interest and its subsequent expression as a fusion protein with an N-terminal 6xHis tag, facilitating purification of the recombinant protein.
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Lab products found in correlation
Pgem t easy vector, by Promega (1 mentions)
Nhs activated hitrap, by GE Healthcare (1 mentions)
Kod plus mutagenesis kit, by Toyobo (1 mentions)
Baculovirus system, by Thermo Fisher Scientific (1 mentions)
Hitrap, by Cytiva (1 mentions)
Bamhi, by Thermo Fisher Scientific (1 mentions)
Ecori, by Thermo Fisher Scientific (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Pfastbac1, by Thermo Fisher Scientific (1 mentions)
X tremegene 9 transfection reagent, by Roche (1 mentions)
6 protocols using pfastbac ht
1
Insect-Optimized RNAi Silencing Genes
2
Purification and Characterization of Akt Constructs
3
Expression and Purification of Autotaxin Isoforms
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Human cDNA for ATXβ was amplified using RT-PCR and human liver cDNA library as template DNA, based on sequence information in a database (GenBank [L46720]). Human ATXβ was introduced into the baculovirus transfer vector pFASTBac-HT, which includes a polyhistidine-tag at the NH2-terminus (Invitrogen, Carlsbad CA). ATXδ (GenBank [AAB00855]), which lacks amino acids 573–576 of human ATXβ (Fig 1 ), was constructed from a plasmid encoding ATXβ using inverted PCR and the KOD-Plus-Mutagenesis Kit (Toyobo, Tokyo, Japan) according to the manufacturer’s instructions, and then introduced to the baculovirus vector. The recombinant polyhistidine-tagged ATXβ (his ATXβ) and polyhistidine-tagged ATXδ (his ATXδ) were produced in Sf9 insect cells with the baculovirus system (Invitrogen, Carlsbad CA) and were purified using chelate column chromatography (BD Talon; BD Biosciences, San Jose, CA) and affinity chromatography with the NHS-activated HiTrap (GE Healthcare, Uppsala, Sweden) immobilized anti-ATX monoclonal antibody R10.7. Using this protocol, ATX could be eluted under mild acidic conditions (100 mmol/L citrate buffer, pH5.0) without the loss of LysoPLD activity.
4
Cloning of Protein into pFastBac HT
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The PCR product was gel purified and double digested with BamHI and EcoRI (Thermo scientific, USA) and then ligated into BamHI and EcoRI pre-digested pFastBac HT (Invitrogen, USA). The ligation product was incubated overnight at room temperature and then used for transformation of E. coli strain Top10. The transformants were plated on LB agar plates containing 100µg/mL ampicillin and incubated at 37 ºC for 24 h.
5
Purification and Characterization of Akt Constructs
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Myc-tagged ACAP1 has been described previously5 (link). Akt forms, wild-type, catalytic dead (K179M), and phosphorylation mutants (T308D/S473D and T308A/S473A) were gifts from Alex Toker (Beth Israel Deaconess Medical Center, Boston, MA). ARNO forms, wild-type and catalytic dead (E156K), in pcDNA3 were gifts from Lorraine Santy (Pennsylvania State University, University Park, PA). The following GST fusion constructs have been described previously: GST-α5 forms (full-length and truncations)13 (link), GST-TfR forms (full-length and truncations)4 (link), and GST-ACAP1 forms (BAR-PH and GAP-ANK)13 (link). GST-Akt forms (full-length and truncations) were subcloned into the BamHI and XhoI sites of the pFastBac™HT plasmid (Invitrogen), followed by baculovirus expression as described above. 6xHis-tagged Akt kinase domain was subcloned into the BamHI sites of the pET15b vector for bacterial expression.
6
Cloning and Expression of FBXO21, EID1, and EID2
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Complementary DNAs encoding FLAG-tagged WT or mutant forms of human FBXO21 were subcloned into pcDNA3 (Invitrogen) or pCSII-CMV (kindly provided by H. Miyoshi), and those for HA-tagged WT or mutant forms of human EID1 or EID2 were subcloned into pcDNA3 (Invitrogen). The resulting vectors were introduced into HEK293T or HeLa cells with the use of the X-tremeGENE9 transfection reagent (Roche, Indianapolis, IN, USA). For baculoviral expression, cDNAs for FLAG-tagged human FBXO21, Myc epitope-tagged mouse CUL1 and SKP1, and an HA-tagged NH 2 -terminal fragment of human EID1 were subcloned into pFASTBacHT (encoding a His 6 tag) (Invitrogen), whereas that for human RBX1 containing an NH 2 -terminal, Myc epitope tag was subcloned into pFASTBac1 (Invitrogen).
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