• RTS E. coli System
• RTS E. coli Technology
• RTS 100 E. coli HY Kit
• RTS 100 E. coli Disulfide Kit
• Using the RTS 100 E. coli System
• RTS 500 ProteoMaster E. coli HY Kit
• RTS 500 E. coli HY Kit
• RTS 500 E. coli Disulfide Kit
• RTS 9000 E. coli HY Kit
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Expression of: The human amyloid protein (13.5 kD - A). A human nuclease (34.6 kD - B) in RTS 100 E. coli HY.
wt: Original DNA sequence was used for protein expression; 1-10: Optimized variants, suggested by ProteoExpert were used for protein expression.
 
Get your proteins active

• Express disulfide-bonded proteins in a functional form under optimized lysate and reaction conditions, provided by the RTS 100 E. coli Disulfide kit.

• Obtain high yields of disulfide-bonded proteins due to the kit’s enhanced biochemistry.

Label proteins for X-ray and NMR structural studies
Specifically label your protein in a small, 1ml reaction volume, by performing the expression in an RTS E. coli kit together with the RTS Amino Acid Sampler, and reduce the metabolic scrambling typically observed with cell-based labeling systems.

The RTS E. coli Kits use HY Technology to reach unprecedented yields in 4-24 hour/s reactions. By reaching as much as 20 µg per 50 µl reaction (RTS 100 E. coli HY Kit), you can perform most analytical assays.
 
The RTS 100 E coli HY Kit is capable of using either circular or linear DNA templates. Experimental designs can take advantage of the Overlap Extension Technology which is the basic principle of the RTS E. coli Linear Template Generation Sets. These sets are used to add the 5' and 3' T7 regulatory regions required for expression in the RTS 100 E. coli HY Kit. They eliminate the subcloning normally required to express proteins.
 
Since the RTS 100 E. coli Disulfide Kit combines HY technology and CECF principle, it can only be used with circular templates. Due to the longer reaction time when using the CECF technology, higher yields of protein are obtained: up to 80 µg / 50 µl (Urokinase). Likewise, the RTS 500 and RTS 9000 kits are performed under HY and CECF conditions. Therefore, cloning of the target sequence in expression vectors suitable for RTS (e.g. pIVEX) is required. Using linear templates is not recommended.     

Product Description
 
The RTS 100 E. coli Disulfide Kit provides an optimized combination of lysate, reagents, chaperones, and buffer systems to produce maximum yields of active multiple-disulfide-bonded protein in a CECF (continuous-exchange, cell-free) system.
 
The Kit employs several innovative technologies:

• Coupled transcription/translation system; no mRNA isolation necessary
• Optimized high-yield E. coli lysate
• Redox buffer to maintain the system under oxidizing conditions, which enhances disulfide-bond formation
• Disulfide isomerase for rearrangement of disulfide bonds, which enhances yields of correctly folded proteins
• Increased amounts of GroE chaperones, which produce higher yields of soluble, properly folded proteins

It utilizes continuous-exchange, cell-free (CECF) technology, which prolongs the reaction time for at least 24 hours and therefore enhances the yield of produced proteins. The kit contains reagents for 24 reactions (50 µl maximum volume each) that are run separately. By combining two kits, 48 reactions can be run simultaneously using the RTS ProteoMaster Instrument.
Transcription, translation, and disulfide-bond formation take place in the 50 µl reaction compartment of the reaction device. Substrates and energy components needed for a sustained reaction are continuously supplied via a semipermeable membrane. At the same time, potentially inhibitory reaction by-products are diluted via diffusion through the same membrane into the 1 ml feeding chamber (see picture below). Depending on the particular protein, expression of up to 50 µg protein per 50 µl reaction can be obtained.
 
Benefits

• Express disulfide-bonded proteins in functional form by using the kit's optimized lysate and reaction conditions.
• Obtain high yields of disulfide-bonded proteins due to the kit's enhanced biochemistry.
• Achieve scalable and preparative cell-free protein expression, with higher yields than in batch reactions.
• Enable expression of toxic proteins that cannot be produced in cellular or bacterial expression systems.
• Express multiple proteins in parallel.
• Incorporate labeled methionine into synthesized proteins.
• Obtain excellent reproducibility.
• Maximize convenience – no fermentation, no safety regulations related to recombinant organisms.

 __

Application of the RTS 100 E. coli Disulfide kit
 
The RTS disulfide system is the only in vitro protein expression system that can express properly folded disulfide-bonded proteins that cannot be produced under reducing conditions. The RTS 100 E. coli Disulfide Kit is designed for cell-free high-throughput expression of disulfide-bonded proteins from plasmid templates. Use the kit for:

• Rapid parallel synthesis of disulfide-bonded proteins
• Rapid functional testing of different constructs (e.g., for epitope or functional domain mapping)
• Expression of toxic gene products
• Production of proteins in a wide molecular weight range
• Optimization of expression conditions (chaperones, detergents, temperature, reaction time, etc.).

For results please go to Literature and Results > Results > RTS 100 E. coli Disulfide Results

• It is recommended to optimize DNA template sequence for high protein yield with the web-based software tool ProteoExpert RTS E. coli HY, especially when eukaryotic proteins are to be expressed in E. coli lysates.
• Optimal performance of the RTS 100 can be achieved using the RTS ProteoMaster Instrument. The instrument provides optimal temperature and shaking conditions for maximal yield of functional protein
• All reagents and devices required for an RTS 100 reaction are provided with the kit. Performing an RTS 100 reaction is simple:
  
  1.  Just add RTS 100 reagents to a microfuge tube or microplate along with the appropriate reagents and your expression construct.
  2. Insert the tube into the RTS ProteoMaster Instrument and start the reaction by pressing the start button
  3. Once the reaction is complete (4 h), the expressed protein can be purified using standard affinity chromatography.
  
  
• After establishing the protein expression conditions in the RTS 100 E. coli, the expression can be scaled up with the RTS 500 formats for yields of up to 6 mg protein / ml without further optimization.

Large Scale Production

IMPORTANT NOTE: The RTS 500 ProteoMaster Device does not fit into the discontinued RTS 500 Instrument.
 
Application of the RTS 500 ProteoMaster E. coli500 HY kit
 
The RTS 500 ProteoMaster E. coli HY kit is designed for

• the expression of proteins in the range of 0.5 - 6 mg/ml, after expression conditions have been optimized with the RTS 100 E. coli HY kit and ProteoExpert E. coli HY.
• In order to produce protein amounts of up to 50 mg / reaction, proceed to the RTS 9000 E. coli HY kit without further optimization.

The RTS 500 E. coli HY Kit - expression of 500 µg to 6 mg of protein/ml in the RTS 500 Instrument*
 
The RTS 500 E. coli HY Kit was initially designed and optimized for the expression of protein using the RTS 500 Instrument*. The kit utilizes the biochemically enhanced HY E. coli lysate and the RTS 500 CECF-reaction device, to obtain up to 6 mg of protein in a 1 ml reaction. The RTS 500 Reaction Device includes two magnetic stirr bars to support CECF reactions. Through stirring via the magnetic bars, the RTS 500 Instrument* guarantees an optimized CECF-process during RTS 500 reactions.
It is possible to use the kit in combination with the RTS ProteoMaster Instrument as well, however, the expression yield can be significantly lower compared to yields obtained in the RTS 500 Instrument*.
 
Two sizes of the kit contain all reagents and reaction devices for either 2 or 5 reactions. pIVEX expression vectors are available separately.

Product Description
 
The RTS 500 E. coli Disulfide Kit provides an optimized combination of lysate, reagents, chaperones, and buffer systems to produce maximum yields of active disulfide-bonded protein in a CECF (continuous-exchange, cell-free) system.
 
The kit employs several innovative technologies:

• Coupled transcription/translation system; no mRNA isolation necessary
• Optimized high-yield E. coli lysate
• Redox buffer to maintain the system under oxidizing conditions, which enhances disulfide-bond formation
• Disulfide isomerase for rearrangement of disulfide bonds, which enhances yields of correctly folded proteins
• Increased amounts of GroE chaperones, which produce higher yields of soluble, properly folded proteins

It utilizes continuous-exchange, cell-free (CECF) technology, which prolongs the reaction time for at least 24 hours and therefore enhances the yield of produced proteins. The kit contains reagents for 5 reactions (1 ml reaction volume each), which can be run simultaneously in the RTS ProteoMaster Instrument.
For reaction devides see RTS 500 ProteoMaster E. Coli Kit.
 
 
Benefits

• Express disulfide-bonded proteins in functional form by using the kit’s optimized lysate and reaction conditions.
• Obtain high yields of disulfide-bonded proteins due to the kit’s enhanced biochemistry.
• Achieve scalable and preparative cell-free protein expression, with higher yields than in batch reactions.
• Enable expression of toxic proteins that cannot be produced in cellular or bacterial expression systems.
• Express multiple proteins in parallel.
• Incorporate labeled methionine into synthesized proteins.
• Obtain excellent reproducibility.
• Maximize convenience – no fermentation, no safety regulations related to recombinant organisms.

 

Application of the RTS 500 E. coli Disulfide Kit
 
The RTS disulfide system is the only in vitro protein expression system that can express properly folded disulfide-bonded proteins that cannot be produced under reducing conditions. The RTS 500 E. coli Disulfide Kit is designed for cell-free expression of disulfide-bonded proteins from plasmid templates. Use the kit for:

• High yield parallel synthesis of disulfide-bonded proteins
• Rapid functional testing of different constructs (e.g., for epitope or functional domain mapping)
• Expression of toxic gene products
• Production of proteins in a wide molecular weight range.

For results please go to Literature and Results > Results > RTS 500 E. coli Disulfide Results

The RTS 9000 E. coli HY Kit and the RTS 9000 Device
 
Application of the RTS 9000 E. coli HY kit
 
The RTS 9000 E. coli HY kit is designed for

• proteins that express at low levels
• generation of sufficient amounts of protein for applications like x-ray crystallography, preparation of antibodies toxicological studies.