• 易锦生物提供的所有人、小鼠和大鼠ORF 克隆及其慢病毒、AAV 颗粒与重组蛋白均使用基于毛细管电泳的 Sanger 法“金标准”全长测序,保证氨基酸序列与NCBI 数据库一致。
    All human and mice ORF clones and related Lentivirus, AAV particles and recombinant proteins provided by iGeneBio are fully sequenced using Sanger sequencing by capillary electrophoresis and Amino Acid Sequences are guaranteed to be matched with NCBI database.
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RTS 100 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 100 Wheat Germ CECF Kit is designed for high-throughput protein expression. It enables high success rates for expression and solubility, particularly for eukaryotic target proteins.
Convenience is greatly improved, due to the use of 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).
Note:  For use with the RTS ProteoMaster Instrument or Eppendorf Thermomixer!

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 100 Wheat Germ CECF Kit, transcription and translation take place simultaneously in the 50 µl reaction compartments 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 (Figure 1). Depending on the protein, expression of up to 20 – 30 µg protein per reaction (with control protein glucuronidase) can be obtained within 24 hours in a 50 µl protein synthesis reaction. Yields may reach up to several hundred micrograms per milliliter when using PCR-generated linear templates, and up to milligram quantities per milliliter of protein-synthesis reaction using vectors or mRNA.

Figure 1: RTS Wheat Germ Lysate-based reactions utilize a specially designed two-chamber microplate device that fits into the RTS ProteoMaster Instrument or Eppendorf Thermomixer.

Background Information

Gene expression in the RTS 100 Wheat Germ CECF Kit can be initiated either by utilizing linear templates generated through PCR using the RTS Wheat Germ Linear Template Generation Set, His6tag 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 for the RTS 100 Wheat Germ CECF Kit).

Note:   We recommend starting with PCR products as expression templates, using the RTS Wheat Germ Linear Template Generation Set, His6-tag. With this approach, multiple constructs can be rapidly generated and screened for yield and solubility in parallel.
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 100 Wheat Germ CECF kit requires the use of the RTS ProteoMaster Instrument or the Eppendorf Thermomixer.

Contents

Each kit contains sufficient reagents for 24 (50 µl) coupled transcription-translation reactions.

  1. Wheat Germ Lysate
  2. Reaction Mix
  3. Feeding Mix
  4. Amino Acids
  5. Methionine
  6. Reconstitution Buffer
  7. GUS (glucuronidase) Control Vector
  8. Reaction Devices (3 disposable two-chamber devices, each for eight continuous-exchange, cell-free (CECF) protein expression reactions, including adhesive film and mounting frame)   
  9. Three spare caps for bottle 3 (Feeding Mix)

Typical Experiment

Figure 2: Expression of SRC kinase. SRC kinase was cloned into pIVEX 1.4 WG (N-terminal His6-tag) vector and expressed with the RTS 100 Wheat Germ CECF Kit. After 3, 6, 12, and 24 hours of expression (lanes 5 – 8), 0.5 µl samples of 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 Chloramphenicol Acetyltransferase (CAT) protein as marker.

Figure 3: Western blot analysis of SRC kinase expression in the RTS 100 Wheat Germ CECF Kit. After 3, 6, 12, and 24 hours of expression, 0.5 µl samples of expression reactions were analyzed by SDS-PAGE then stained with an anti-His6-peroxidase antibody.

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