Recombinant Protein Expression

RTS 500 Wheat Germ CECF Kit

Application

The RTS Wheat Germ product line combines several new technologies for efficient and optimized protein expression using a eukaryotic cell lysate. It overcomes limitations that often restrict the use of E. coli-based systems, especially in the production of eukaryotic target proteins.
The RTS 500 Wheat Germ CECF Kit enables high success rates for expression and solubility, particularly for eukaryotic target proteins. Convenience is greatly improved, due to the ability to use PCR-generated linear templates; plasmids and mRNA can also be used as template. Use this kit to synthesize proteins in a wide molecular weight range (up to 220 kD, as tested) as well as to synthesize truncated protein variants (e.g., for epitope or functional domain mapping).

Benefits

  • Achieve scalable and preparative eukaryotic cell-free protein expression.
  • Express eukaryotic proteins with high success rates, generally without sequence optimization.
  • Maximize the production of active heat-sensitive proteins, due to an optimum reaction temperature of 24°C.
  • Avoid cloning; express your protein directly from PCR products; or utilize vectors or mRNA as template.
  • Enable expression of toxic proteins that cannot be produced in cellular or bacterial expression systems.
  • Express multiple proteins in parallel.
  • Incorporate labeled methionine or selenomethionine into synthesized proteins.
  • Obtain excellent reproducibility.
  • Maximize convenience; no fermentation, no safety regulations related to recombinant organisms.

Product Description

In contrast to traditional eukaryotic in vitro transcription/translation reactions, which run for only 4 hours in batch mode, the RTS Wheat Germ system is the only eukaryotic protein synthesis system that uses the continuous-exchange, cell-free (CECF) technology. This CECF system prolongs the reaction for at least 24 hours and increases the yield dramatically. In the RTS 500 Wheat Germ CECF Kit, transcription and translation take place simultaneously in the 1 ml reaction compartment of the reaction device. Substrates and energy components needed for a sustained reaction are continuously supplied via two semipermeable membranes. At the same time, potentially inhibitory reaction by-products are diluted via diffusion through the same membranes into the 10 ml feeding chamber (Figure 1).
The system accepts linear templates as well as mRNA and vectors/plasmid-DNA. Depending on the protein, expression yields of more than 1 mg protein per reaction can be obtained within 24 hours in a 1 ml protein synthesis reaction.




Figure 1: RTS 500 reaction device. The disposable device has three major parts: a 1-ml vertical reaction compartment, two semipermeable membranes, and a 10-ml feeding compartment.

Background Information

Gene expression in the RTS 500 Wheat Germ CECF system can be initiated either by utilizing linear templates generated through PCR using the RTS Wheat Germ Linear Template Generation Set, His6-tag, or via standard cloning using the RTS pIVEX Wheat Germ His6-tag Vector Set. mRNA obtained by in vitro transcription can also be used for protein expression (see the pack insert of the RTS 500 Wheat Germ CECF Kit).
Common limitations of RTS Wheat Germ lysates: The RTS Wheat Germ system (in the absence of further modification) cannot perform the following post-translational modifications of the expressed proteins:
  • Glycosylation
  • Disulfide-bond formation
  • Signal sequence cleavage

The RTS 500 Wheat Germ CECF Kit requires the use of the RTS ProteoMaster Instrument or the Eppendorf Thermomixer in combination with the RTS 500 Adapter!

Contents

Each kit contains enough reagents for 5 coupled transcription-translation or translation-only reactions of 1 ml.
  1. Wheat Germ Lysate
  2. Reaction Mix
  3. Feeding Mix
  4. Amino Acids
  5. Methionine
  6. Reconstitution Buffer
  7. Control Vector Glucuronidase
  8. Five Reaction Devices
  9. Five spare caps for bottle 3 (Feeding Mix)

Typical Experiment



Figure 2: Expression of Steroid Hormone Receptor (SHR). SHR was cloned into the pIVEX 1.4 WG (N-terminal His6-tag) vector and expressed with the RTS 500 Wheat Germ CECF Kit. After 3, 6, 12, and 24 hours of expression (lanes 5 - 8), 0.5 µl samples of the expression reactions were analyzed by SDS-PAGE, and the gel was stained with Coomassie Brilliant Blue. Lane 1: NC = negative control Lanes 2 - 4:  400, 500, and 700 ng/lane of Bovine Serum Albumin (BSA) as a marker



Figure 3: Western blot analysis of Steroid Hormone Receptor (SHR) expression in the RTS 500 Wheat Germ CECF Kit. After 3, 6, 12, and 24 hours of expression, 0.5 µl of expression reactions were analyzed by SDS-PAGE and stained with an anti-His6-peroxidase antibody.

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