Human Cell-Free Protein Expression System and vectors
The Human Cell-Free Protein Expression System (Cat. # 3281) 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.
*Product availability varies by region.
The Human Cell-Free Protein Expression System (Cat. # 3281) 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.
*Product availability varies by region.
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 in in vivo systems can be easily expressed in vitro.
Bulk sizes are available for high-throughput studies.
Overview
- 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 in vitro translation systems
- Works well with challenging applications such as large proteins (over 150 kDa)
- Amenable to high-throughput screening; bulk sizes are available
More Information
Applications
- 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 that are easily degraded or insoluble in conventional in vivo expression systems such as E. coli
- High-throughput proteomic studies
Storage
- 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
Components
| Human Cell-Free Protein Expression System | |
|---|---|
| Cell Lysate | 100 µl |
| Mixture-1 | 60 µl |
| Mixture-2 | 10 µl |
| Mixture-3 | 20 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 |
Notes:
- Dissolve Cell Lysate just prior to use, mixing gently by pipetting. Use immediately. After use, promptly store at –80°C. Although up to five freeze-thaw cycles should not affect performance, store the Cell Lysate in small-volume, ready-to-use 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.
pT7-IRES vectors
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 |
Notes:
- 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)
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. Availability of products in the In-Fusion portfolio varies by geographic location; see the In-Fusion product pages for more information about products available in your region.
Additional product information
Please see the product's Certificate of Analysis and User Manual for information about storage conditions, product components, and technical specifications. Technical documents are located under the Documents tab.
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