pEXP4-DEST Vector Kit

Catalog number: V96004

Invitrogen™  Related applications: Gateway Cloning | Protein Expression

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Description

Expressway™ Tag-On-Demand™ Cell-Free E. coliExpression System with Lumio™ Technology enables convenient, in vitro expression of tagged or untagged protein from a single vector, real-time detection of protein synthesis, and easy in-gel protein detection. With this system, you can:

• Quickly screen for expression levels of full-length protein
• Save time and effort by cloning only once to express tagged or native protein
• Express a C-terminally tagged protein from an Ultimate™ ORF Clone in an in vitro system

The Expressway™ Tag-On-Demand™ System includes a specialized E. colilysate derived from a slyD mutant for cell-free synthesis. This lysate eliminates non-specific binding of the Lumio™ Green Detection Reagent to the endogenous SlyD protein, providing an optimal background for recombinant protein detection.

How it works
Simply clone your gene of interest containing a TAG (amber) stop codon into the provided pEXP4-DEST (Figure 1). The pEXP4-DEST vector features attR sites for efficient Gateway® recombination, C-terminal Lumio™ and 6xHis tags for simplified identification and purification, and components for cell-free protein synthesis. In the presence of the Expressway™ Tag-On-Demand™ Suppressor tRNA, the TAG codon is not recognized and translation continues through the Lumio™ and 6xHis tags. In the absence of the Suppressor tRNA, the TAG stop codon is recognized and native protein is produced (Figure 2). From the same vector, you can rapidly determine the expression level of the C-terminal-tagged, full-length protein, and then express the native protein free from any adverse affects the tag may have on its structure, activity, or molecular interactions.
For Research Use Only. Not for use in diagnostic procedures.

Specifications

Promoter: T7
Vector Type: pEXP, pDEST
Product Size: 6 µg
Cloning Method: Gateway®
Protein Tag or Fusion: Lumio
Selection Agent (Eukaryotic): None
Antibiotic Resistance (Bacterial): Ampicillin (AmpR), Zeocin™ (ZeoR)

Contents & storage

The Expressway™ Tag-On-Demand™ Cell-Free E. coli Expression System with Lumio™ Technology includes pEXP4-DEST vector (when indicated), positive expression control plasmid, Tag-On-Demand™ slyD Mutant E. coli Extract, E. coli Reaction Buffer, Tag-On-Demand™ Suppressor tRNA, Methionine, DNase/RNase-free distilled water, RNase A, T7 Enzyme Mix, 2-ml reaction tubes, Lumio™ Green Detection Kit, and the Benchmark™ Fluorescent Protein Standard. The pEXP4-DEST Gateway® Vector Kit includes pEXP4-DEST and a control plasmid. Store vectors, detection reagent, and protein standard at -20°C. Store all other components at -80°C. Guaranteed stable for 6 months when properly stored.

Documents

Manuals & protocols