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Gibco™

Bac-to-Bac™ HBM TOPO™ Cloning Kit

Catalog number: A11338
Gibco™

Bac-to-Bac™ HBM TOPO™ Cloning Kit

Catalog number: A11338
Catalog Number
A11338
Unit Size
20 reactions
Price (USD)
Catalog NumberUnit SizePrice (USD)
A1133820 reactionsContact Us ›
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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.
For Research Use Only. Not for use in diagnostic procedures.

Specifications

Cell Line
High Five™, Sf21, Sf9
Cell Type
Insect Cell
Cloning Method
Blunt TOPO
Cleavage
TEV Protease Recognition Site
Format
Kit
Expression Mechanism
Cell-Based Expression
Expression System
Baculovirus
For Use With (Application)
Protein Expression
High-throughput Compatibility
High-throughput Compatible, Not High-throughput Compatible (Manual)
Key Functions
Secreted Expression
No. of Reactions
20 Reactions
Product Line
Bac-to-Bac™, TOPO™
Product Type
Cloning Kit
Promoter
Polyhedrin
Protein Tag
HBM (Honeybee Melittin), His Tag (6x)
Quantity
20 Reactions
Selection Agent (Eukaryotic)
None
Vector
pFastBac/HBM-TOPO

Contents & Storage

Bac-to-Bac™ HBM TOPO™ Cloning Kit contains: (For details see the manual)
• Vector kit for 20 topo cloning reactions
• pFastBac⁄HBM TOPO™ Vector containing the C-terminal TEV cleavage sit and His-Tag.
• pFastBac⁄CT-Gus Control Plasmid (Control expression vector)
• 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):
• 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

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