Bac-to-Bac™ Baculovirus Expression is an efficient method for producing baculovirus for high-level protein expression in insect cells. It relies on generation of recombinant virus by site-specific transposition in E. coli rather than homologous recombination in insect cells. New: pFastBacTOPO™ vectors!
Reliable and fast protein expression The Bac-to-Bac™ Baculovirus Expression System is faster and easier than traditional baculovirus expression, and it maintains high levels of protein expression. Bac-to-Bac™ relies on generation of recombinant baculovirus by site-specific transposition in E. coli rather than homologous recombination in insect cells. The Bac-to-Bac™ Baculvirus Expression System is highly regarded in academic Literature. Approximately 100 citations every year since 2000!
Traditional baculovirus systems require purification and amplification of an initial low-titer viral supernatant. This requires a time-consuming plaque assay. Bac-to-Bac™'s pFastBac™ vector, however, recombines with the parent bacmid in DH10Bac™ E. coli competent cells to form an expression bacmid. Transfect the bacmid into insect cells for fast production of a high titer of pure recombinant baculovirus particles in the very first transfection. You'll save weeks of precious time. Collect a pure P2 baculovirus stock on Day 10 without the necessity of tedious plaques assay (See Figure 1)
High expression, easy screening pFastBac™ uses the strong polyhedrin promoter to generate high levels of expression in a variety of insect cell lines such as Sf9, Sf21, and High Five™ cells.
New additions: Bac-to-Bac™'s pFastBac vectors are now available as pFastBacTOPO™ vectors too. pFastBacTOPO™ vectors are Blunt™ TOPO™ vectors and are supplied with Mach1™-T1R E. coli for easy cloning and pFastBac⁄GOI propagation. The new vectors enable you to:
Amplify your gene of interest (GOI) with a PCR enzyme of highest fidelity such as Accuprime™ Pfx SuperMix.
TOPO™ clone the blunt-end PCR product into the new vector in only 5 minutes!
Visualize colonies 8 hours after plating on ampicillin selective plates because Mach1™-T1R E. coli cells have a faster doubling time compared to other standard cloning strains.
With the Bac-to-Bac™ C-His TOPO™ cloning or expression kit, you can produce a C-terminal His-fusion protein with a TEV cleavage site to purify with nickel-chelating resins (Invitrogen’s ProBond™ Purification System) and generate a native protein with the aid of Invitrogen’s popular AcTEV™ Protease. This is the Invitrogen’s only vector that contains a C-terminal TEV cleavage site!
The new vectors combine the TOPO™ cloning technology with the highly regarded Bac-to-Bac™ baculovirus expression features to enable easy cloning and high-level protein expression. The pFastBacTOPO™ vectors are offered as cloning and expression kits.
Kit content for Bac-to-Bac™ C-His TOPO™ cloning kit (For details see the manual) • Vector kit for 20 topo cloning reactions: o pFastBac⁄CT-TOPO™ Vector containing the C-terminal TEV cleavage sit and His-Tag. o pFastBac⁄CT-Gus Control Plasmid (Control expression vector) o Other reagents supplied: 10x PCR, dNTP Mix, Salt Solution, Sterile Water, Control PCR template, Polyhedrin Forward Primer, SV40 pA Reverse Primer • Kit containing competent cells (20 reactions): o One Shot™ Mach1-T1R Chemically Competent E. coli
Vector kit: store at -20°C One Shot™ Mach1-T1R Chemically Competent E. coli: store at -80°C
