Clone a PCR amplified gene in an effortless afternoon, and express recombinant protein the next day.
Complete Cloning and Expression Systems with Expressioneering™ Technology
The Expresso Rhamnose Cloning and Protein Expression Systems are designed for fast, easy, and efficient directional cloning and expression of PCR-amplified genes using Expressioneering Technology. Expressioneering Technology uses in vivo homologous recombination to seamlessly clone PCR amplified DNA into specially designed expression vectors without the need for enzymes or purification steps. A single host strain is used for both stable cloning and controlled protein expression, making Expresso Rhamnose the fastest cloning and expression systems available. The systems come complete with pre-processed expression plasmids and competent cells, supplied in single transformation vials.
Figure 1. Expressioneering Technology uses in vivo homologous recombination to seamlessly clone PCR amplified DNA into specially designed expression vectors without the need for enzymes or purification steps. The desired insert is simply amplified with primers that include 18 bases that overlap with the ends of the Expresso® vector. The unpurified PCR amplicon is then mixed with the pRham expression plasmid and the high-efficiency competent cells provided, and directly plated on appropriate media.
The Expresso Rhamnose Systems utilize the rhamnose-inducible rhaPBAD promoter for tight control of protein expression. Transcription from the rhaPBAD promoter can be “tuned” using different concentrations of rhamnose to identify the optimal expression levels for difficult target proteins. Simple autoinduction protocols using rhamnose and glucose solutions supplied with the kits allow protein expression with minimal intervention.
The pRham™ N-His and pRham C-His Vectors provided in the Expresso Rhamnose Cloning and Expression System facilitate instant cloning of target genes with a choice of amino- or carboxyl-terminal 6xHis affinity tags. The 6xHis peptide provides for fast and easy affinity purification of proteins under native or denaturing conditions. For enhanced soluble protein expression using SUMO fusion tag technology, see the Expresso Rhamnose SUMO System.
With instant cloning using Expressioneering Technology, a single-host system for cloning and expression, and simple autoinduction protocols, the Expresso Rhamnose Systems are highly amenable to high-throughput cloning and protein expression.
Figure 2. pRham expression vectors. RBS, ribosome binding site; ATG, translation start site; Stop, translation end site; Kan, kanamycin resistance gene; ROP, Repressor of Priming (for low copy number); Ori, origin of replication. CloneSmart® transcription terminators (T) prevent transcription into or out of the insert, and a terminator follows the cloning site. The 6xHis affinity tag is fused to the amino terminus (pRham N-His) or at the carboxyl terminus (pRham C-His) of the expressed target protein. Also available: pRham N-His SUMO Vector for enhanced soluble protein expression with cleavable SUMO solubility tag.
pRham Expression Vectors
The pRham™ N-His and pRham C-His Vectors provided in the Expresso Rhamnose Cloning and Expression System are shown in figure 2. Like the pETite Vectors featured in the Expresso T7 kits, the pRham Vectors used in the Expresso Rhamnose kits are based on the pSmart vector backbone, which features patented CloneSmart® technology for increased cloning efficiency. The pre-processed pETite and pRham vectors feature the same sequences flanking the cloning site, allowing a single PCR product to be cloned into either vector.
Single-Host cloning and tunable expression with Expresso Rhamnose System
Figure 3. Tuning recombinant protein expression levels with rhamnose induction. The pRham C-His Kan Vector containing a gene encoding a blue fluorescent protein (BFP) was transformed into E. cloni 10G cells. An uninduced starter culture was inoculated to a starting OD600 of 0.8 into culture tubes containing LB media with 30 µg/ml kanamycin and the indicated concentrations of rhamnose (0 to 0.2% w/v) or 2% glucose. After overnight incubation at 37°C, samples were harvested by centrifugation, lysed in SDS-PAGE loading buffer, and analyzed by SDS-PAGE. The Coomassie-blue stained gel shows total cellular protein. Protein expression levels are responsive to rhamnose concentrations between 0.001% and 0.2%.
The rhaPBAD promoter is tightly shut off in the absence of the sugar rhamnose, allowing the use of a single host strain for both cloning and protein expression. The level of induction from rhaPBAD is responsive to different concentrations of rhamnose (Fig. 3), allowing you to tune the level of expression, especially for proteins that are toxic or insoluble when overexpressed. Maximal protein expression from the rhaPBAD promoter is typically lower than from the T7 promoter, but can still reach very high levels (up to 100 mg/l ).
Figure 4. Autoinduction of protein expression with the Expresso Rhamnose System. Flasks of LB media containing 30 µg/ml kanamycin, 0.2% rhamnose, and either 0.05% glucose (early autoinduction, upper panel) or 0.15% glucose (late autoinduction, lower panel) were inoculated to an initial OD600 of 0.4 with an uninduced culture of E. cloni 10G cells harboring the T4 ligase gene in the pRham N-His Vector. Samples of the cultures were harvested at the indicated time points for SDS-PAGE analysis. Induction of ligase expression began by 4 hours in the early autoinduction culture, or 8 hours in the late autoinduction culture. Both cultures reached similar OD600 by 24 hours.
Figure 5. Direct autoinduction of recombinant protein expression from individual colonies. The pRham C-His Vector containing the BFP gene was transformed into E. cloni 10G cells and plated on YT agar plates containing 30 µg/ml kanamycin. Single colonies were picked from the plate and inoculated directly into LB liquid media containing kanamycin (30 µg/ml), rhamnose (0.2% w/v), and either 0.05% (early autoinduction) or 0.15% (late autoinduction) glucose. Samples were harvested at the time points indicated and analyzed by SDS-PAGE.
Convenient Autoinduction with Expresso Rhamnose Systems
Transcription from the rhaPBAD promoter is subject to repression by glucose. When both glucose and rhamnose are present, glucose is metabolized preferentially and the rhaPBAD promoter remains inactive. Upon depletion of glucose, the rhaPBAD promoter is activated by rhamnose. Convenient autoinduction protocols use a combination of glucose and rhamnose to allow induction of protein expression with minimal intervention. Inoculate from a starter culture (Fig. 4) or directly from individual colonies (Fig. 5) into autoinduction media, and induction occurs automatically. The timing of induction can be adjusted with the use of different glucose concentrations. Solutions of rhamnose and glucose are provided with the Expresso Rhamnose kits.
See Application Note in Nature Methods, "Expresso® Cloning and Expression Systems: Expressioneering™ Technology streamlines recombinant protein expression."
Important Product Use Information:
This product is the subject of U.S. Patent #6,709,861. Additional patent applications owned by Lucigen Corporation are pending.
The 6xHis tag is licensed from Hoffmann-La Roche, Inc., Nutley, NJ and/or Hoffmann-LaRoche Ltd., Basel, Switzerland and is provided only for the use in research. Information about licenses for commercial use is available from QIAGEN GmbH, QIAGEN Strasse 1, D-40724 Hilden, Germany. Purification of 6xHis tagged proteins with Ni-NTA manufactured by QIAGEN is highly recommended for best performances and to avoid poor purification results.
SUMO Express Protease is manufactured and supplied by LifeSensors, Inc.
Please note that the SUMO Protease and SUMO Fusion tag are modified from the original native proteins. Native or non-modified control proteins or proteases will not be compatible. For any questions about the SUMO tag or SUMO Protease and associated uses, please contact Lucigen Technical Service.
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