Human Cell-Free Protein Expression System and Vectors
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The Human Cell-Free Protein Expression System (Cat. #3281) from Takara Bio enables simple and easy in vitro transcription and translation. The single-tube reaction format is easily assembled and protein expression can be completed in as little as 1 hour at 32°C. The Human Cell-Free Protein Expression System allows for high-yield expression and purification of functional protein, including modified proteins and large proteins (over 150 kDa). The Human Cell-Free Protein Expression Maxi System (Cat. #3285) further increases recombinant protein expression by utilizing a dialysis technique that continually replenishes ATP and amino acids during protein translation.
Genes of interest may be cloned rapidly into the pT7-IRES vectors using In-Fusion cloning technology. Using the pT7-IRES vectors, recombinant proteins can be tagged with N-terminal or C-terminal His epitope tags or an N-terminal Myc epitope tag.
Cell-free systems for in vitro transcription and in vitro translation offer several advantages for protein expression and purification:
- They allow quick analyses of protein functions or features.
- They are amenable to high-throughput studies.
- Proteins that are degraded, insoluble or toxic using in vivo systems can be easily expressed in vitro.
- In vitro translation systems allow expression of proteins with post-translational modifications.Bulk sizes are available for high-throughput studies.
- Easy-to-use system allows generation of protein in as little as 1 hour in a single-tube reaction.
- Provides higher yield and greater consistency of functional protein compared to rabbit reticulocyte or wheat germ-based in vitro translation systems.
- Works well with challenging applications such as large proteins (over 150 kDa) and proteins requiring post-translational modification.
- Amenable to high-throughput screening; bulk sizes are available.
- Expression of toxic proteins that are lethal for in vivo expression systems
- Quick analysis of protein functionality
- Rapid analysis of protein mutations or truncations
- Expression of proteins with eukaryotic post-translational modifications
- Expression of proteins that are easily degraded or insoluble in conventional in vivoexpression systems such as E. coli
- High-throughput proteomic studies
Human Cell-Free Protein Expression System
|Cell Lysate||100 µL|
|T7 RNA Polymerase (200 U/µL)||10 µL|
|pT7 IRES Vector (0.5 µg/µL)||20 µL|
|Control Vector (0.3 µg/µL)||10 µL|
- Dissolve Cell Lysate just prior to use, mixing gently by pipetting. Use immdiately. After use, promptly store at -80°C. Although up to five freeze-thaw cycles should not affect performance, store the Cell Lysate in small ready-to-use volume aliquots.
- Mixture-2 and Mixture-3 contain protein. To avoid deactivation, do not stir excessively or vortex.
- The Control Vector contains a beta-galactosidase gene cassette.
- The pT7 IRES Vector included in the kit does not encode fusion tags. Vectors encoding a His or c-myc tag are sold separately.
Each vector (Cat. #3290, #3291, #3292) contains 20 µg DNA at a concentration of 0.5 µg/µL (40 µL total volume).
Human Cell-Free Protein Expression Maxi System
|1) Human Cell-Free Protein Expression System (Cat. #3281)||2 kits|
|2) Supplement Buffer (Human Cell-Free Protein Expression)||15 mL|
|3) Dialysis Columns & Accessories||25 sets|
- Items (2) and (3) are not available for sale separately.
- Item (3) is manufactured by G-Biosciences and includes the following components:
- Tube-O-DIALYZER™, Micro 15K MWCO (5 tubes) Note: Each tube is filled with a preservative solution. The shipping package also contains a small amount of preservative solution to maintain moisture.
- Micro Dialysis Cup (5 cups)
- Float for Micro, for Dialysis Cup (5 floats)
- Human Cell-Free Protein Expression System (Cat. # 3281): -80°C
- pT7-IRES Vectors (Cat. #3290, #3291, #3292): -20°C
- Human Cell-Free Protein Expression Maxi System (Cat. #3285): Dialysis columns & accessories 4°C; All other components -80°C
pT7-IRES Vector information
Vectors in the pT7-IRES series include a T7 promoter and EMCV IRES sequence to facilitate transcription and translation in the Human Cell-Free Protein Expression System as well as a Multiple Cloning Site (MCS), convenient tags (N- or C-terminal His or N-terminal Myc tag sequences), a Factor Xa protease cleavage site for tag removal, polyA signal and T7 terminator.
- pT7-IRES His-N DNA (Cat. # 3290) encodes an N-terminal His tag
- pT7-IRES His-C DNA (Cat. # 3291) encodes a C-terminal His tag
- pT7-IRES Myc-N DNA (Cat. # 3292) encodes an N-terminal c-Myc tag
For vector maps and sequence files, see User Manuals and Vector Information, respectively, under “Documents”.
Related In-Fusion Cloning products
For rapid and easy cloning, use the In-Fusion Cloning Systems or Kits. Products in the In-Fusion portfolio vary by geographic location; see the In-Fusion product pages from Clontechfor more information about products available in your region.