CASCADE™ Protein Expression
CASCADE™ is an Escherichia coli-based recombinant protein expression system that employs two salicylate-responsive transcriptional activator proteins, NahR XylS. This is achieved through the stable insertion of a regulatory module into the chromosome. In the presence of inducer (salicylate & 3-methylbenozate), the NahR protein induces expression of XylS from the Psal promoter. Activation of XylS also occurs, which induces high-level expression of the gene of interest from its cognate Pm promoter. The synergistic effect of using two transcriptional regulators in a sequential cascade amplifies expression levels nearly 20-fold compared to expression from the individual promoters.
Advantages:
- Very low basal expression
- High expression levels following induction
- a variety of genetic backgrounds are available (e.g. K12, BL21)
- Easy custom engineering of new expression strains by insertion of the regulatory module
- Scalable protein expression using inexpensive inducers
- Stable production levels under high density culture conditions
CASCADE™ Quick Tips
CASCADE™ is an Escherichia coli-based recombinant protein expression system that employs two salicylate-responsive transcriptional activator proteins, NahR XylS. This is achieved through the stable insertion of a regulatory module into the chromosome. In the presence of inducer (salicylate & 3-methylbenozate), the NahR protein induces expression of XylS from the Psal promoter. Activation of XylS also occurs, which induces high-level expression of the gene of interest from its cognate Pm promoter. The synergistic effect of using two transcriptional regulators in a sequential cascade amplifies expression levels nearly 20-fold compared to expression from the individual promoters.
E.coli REG-12 Expression Strain
REG-12 is a rifampicin-resistant E. coli K12 mutant that contains the regulatory element nahR/Psal::xylS integrated into the chromosome. This strain is a fast growing E. coli that can host the CASCADE expression vectors. REG_12 is capable of reaching high cell density in rich media and can also grow in minimal media with ammonia and glucose as nitrogen and carbon sources, respectively.
QT pALEX2 Expression Vectors
The NEW pALEX2 vector series is designed for use with the CASCADE system for protein expression. pALEX2(A-c) incorporate a mutant Pm promoter with even lower basal activity than previous generations of the pALEX vectors. The improved multiple cloning site 9MCS) allows for easy and flexible cloning in-frame with a variety of fusion proteins. Full access to vector sequences also facilitates the preparation of pALEX2 vectors for igation-independent and recombination-based cloning strategies.
The pALEX2 vectors contain an enterokinase cleavage site for tag removal. Rare-cutting NotI restriction sites facilitate subcloning into the mini Tn5 vectors for construction of genetically stable expression strains.
QT Ordering Information
The pALEX2 vector series, pALEX-Ca-GFP control vector, REG-12 E. coli, and inducer solution are sold together as part of the LYTAG 2-Phase Purification System.
| Component | Volume | Cat. No. | Pricing |
| Top Solution | 20 mL | RS-4751 | |
| Lower Solution | 10 mL | RS-4752 | |
| LYTAG 2-phase wash buffer (2x) | 65 mL | RS-4753 | |
| 3M Choline Chloride | 10 mL | RS-4755 | |
| 1M Inducer Cocktail | 25 mL | RS-3247 | |
| pAlex2 (a-c) | 8 µg ea. | EV-4658 | |
| pALEX2-Ca-GFP positive control | 8 µg | EV-4757 | |
| E. coli REG-12 | stab | BS-3458 | |
| 20 mL Kit | KT-4697 | 1195 USD |
