Invitrogen™ Bac-to-Bac™ HBM TOPO™ Secreted Expression System

Bac-to-Bac™ HBM TOPO™ Secreted Expression System

Supplier:  Invitrogen™ A11339

Catalog No. A11339

Description

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 (DH10Bac™) rather than homologous recombination in insect cells. The new pFastBac™ HBM TOPO™ vector enables secreted protein expression because it has the honeybee melittin (HBM) secretion signal. The new vector should be strongly considered for the expression of glycoproteins. Glycoproteins cannot be glycosylated in the absence of any secretion signal. In contrast to glycoproteins secreted from mammalian cells, glycoproteins secreted from baculovirus can be easily de-glycosylated in vitro. This is a very important feature in order to crystallize proteins!

The pFastBac™ HBM TOPO™ vector has also a C-terminal His-Tag with a TEV cleavage signal to enable easy purification of Histidine fusion proteins on nickel-chelating resins (ProBond™ Purification System) and native proteins with the aid of AcTev™ protease.
The vector uses the strong polyhedrin promoter to generate high levels of expression in a variety of insect cell lines such as Sf9, Mimic™ Sf9, Sf21, and High Five™ cells in Sf-900 II & Sf-900™ III media from Gibco.

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™ vectors, 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.

Specifications

• Type: Pichia Protein Expression System
• Protein Tag: His Tag (6x)
• Content And Storage: Bac-to-Bac HBM TOPO Secreted Expression System:
  • Vector kit for 20 TOPO cloning reactions, store at -20°C
     –pFastBac/HBM-TOPO Vector containing C-terminal TEV cleavage site and His-tag
     –pFastBac 1-Gus control plasmid (control expression vector)
     –Other kit reagents: 10X PCR, dNTP mix, Salt solution, sterile water, control PCR template, polyhedrin forward primer, SV40 pA reverse primer
  • One Shot Mach1-T1R Chemically Competent E. coli for 20 reactions (20 x 50 μL), store at -80°C
  • MAX Efficiency DH10Bac Competent E. coli for 20 reactions (4 kits with 5 x 0.1 mL each), store at -80°C
  • ExpiFectamine Sf Transfection Reagent (1 mL), store at 4°C, do not freeze
  
  
• Expression Mechanism: Cell-Based Expression
• Product Line: Bac-to-Bac™, TOPO™

• Cloning Method: Blunt TOPO™
• Quantity: 20 Reactions
• Selection Agent (Eukaryotic): None
• Expression System: Baculovirus
• Promoter: Polyhedrin

For Research Use Only. Not for use in diagnostic procedures.