pEXP4-DEST Vector Kit
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Invitrogen™

pEXP4-DEST Vector Kit

Expressway™ Tag-On-Demand™ Cell-Free E. coliExpression System with Lumio™ Technology enables convenient, in vitro expression of tagged or untagged protein fromRead more
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Catalog number V96004
Price (USD)
636.00
Each
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Price (USD)
636.00
Each
Add to cart
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
PromoterT7
Product TypeGateway System Destination Expression Vector
Selection Agent (Eukaryotic)None
Antibiotic Resistance BacterialAmpicillin (AmpR), Zeocin™ (ZeoR)
Protein TagLumio
Cloning MethodGateway
Quantity6 μg
VectorpEXP, pDEST
Product LineExpressway™, Gateway™
Unit SizeEach
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.

Frequently asked questions (FAQs)

I accidentally stored my E. coli slyD-Extract, E. coli Reaction Buffer (-A.A.), and 2X feed buffer at room temperature. Can I still use them?

Unfortunately, this may result in a loss of activity.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I've run out of the T7 RNA polymerase for my cell-free expression. What do you suggest I use?

We would recommend using T7 RNA polymerase (Cat. No. 18033019, 50 U/µL). Use 1-1.5 µL in a 50 µL reaction system.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm getting smearing after running my cell-free expression reaction on a gel.. What could be the cause of this?

Smearing may occur if samples for the following reasons:

- Samples were not precipitated with acetone: precipitate proteins with acetone to remove background smearing.
- Too much protein was loaded: reduce the amount used.
- The gel itself was not clean: rinse the gel briefly before exposing to film.
- Ethanol was present in the protein synthesis reaction: make sure that any residual ethanol is removed during DNA purification.
- Check the date of your pre-cast gels: do not use gels after the expiration date.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm seeing a ladder of small-sized products after running my reaction on a gel when using the Expressway system. Why is this?

There may be several reasons for why this is occurring. The most common are: proteolysis, degradation of DNA and/or RNA templates (truncated templates will generate truncated protein products), internal initiation (if there are many methionines and internal RBS-like sequences in the gene, the ribosome may initiate translation from the wrong methionine), premature termination, translational pausing, frequent rare codon usage, complicated secondary structure of RNA, and others. This can also happen if proteins are denatured for too long, or not enough SDS was added to the 1X SDS-PAGE sample buffer.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

With a cell-free expression system, I'm getting good protein yield, but it has low biological activity. What can I do?

- Your protein may not be folding properly: try to reduce the incubation temperature to as low as 25 degrees C during synthesis.
- You may require post-translational modification of your protein: the Expressway system will not introduce post-translational modifications to the recombinant protein.
- Your synthetic protein may require co-factors for complete activity: try adding required co-factors to the protein synthesis reaction.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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