Description
This is an engineered T7 RNA polymerase variant (low dsRNA) derived from the wild-type T7 RNA polymerase and produced in Escherichia coli. It significantly reduces the production of double-stranded RNA (dsRNA) while efficiently incorporating cap analogs, and exhibiting highly efficient in vitro transcription (IVT) comparable to the wild-type T7 RNA polymerase. It catalyzes the 5'→3' synthesis of RNA on double-stranded DNA from its T7 promoter sequence (5'-TAATACGACTCACTATAG*-3') and uses NTPs as substrates.
Note: G* is the first base of the RNA transcript.
Feature
- dsRNA level lower to about 1/100000
- Comptiable with Trilink CleanCap AG
- High yields comparable to WT
- Lower Cap input
- Animal origin-free (AOF)
Please find information on the development of this enzyme.
Components
|
Components No. |
Name |
10628ES10 |
10628ES60 |
10628ES72 |
10628ES86 |
|
|
|
(10 KU) |
(100 KU) |
(250 KU) |
(2,500 KU) |
|
10628 |
CleaScrip™ T7 RNA Polymerase (low dsRNA, 250 U/μL) |
40 μL |
400 μL |
1 mL |
10 mL |
Specifications
|
Source |
Recombinant E. coli with T7 RNA Polymerase gene |
|
Optimum Temperature |
37℃ |
|
Storage Buffer |
50 mM Tris-HCl, 1 mM EDTA, 10 mM DTT, 100 mM NaCl, 0.1% Triton X-100，50% (v/v) glycerin，pH7.9 at 25℃ |
|
Unit Definition |
The amount of enzyme required to incorporate 1 nmol of [3H] GMP into the acid-insoluble precipitate within 1 hour at 37°C and pH 8.0 is defined as 1 unit. |
|
Recommended Mg2+ |
30mM Magnesium Acetate |
QC Standard
|
Items |
Specification/Standard |
|
Enzyme activity |
250-300U/μL |
|
Protein Purity |
≥95% |
|
Endotoxin |
<20 EU/mg |
|
Protease |
Negative |
|
Exonuclease |
Negative |
|
Nickase |
Negative |
|
RNase |
Negative |
|
E.coli host protein |
<50ppm |
|
E.coli host DNA |
<10 fg/U |
|
Mycoplasma examination |
Negative |
Related Products
Tris NTPs synergistically decreasing dsRNA
Figures
Figure 2. Evaluation of the immunogenicity of the IVT products in murine RAW264.7 cells (Figures 2A and 2B). IFN-β mRNA and protein levels were reduced in RAW264.7 cells transfected with mRNA produced by mutants compared to wild-type, indicating that mRNA synthesized by the wild-type T7 RNAP elicited the strongest immune response, while mRNA from the mutants showed a significantly reduced response.
Figure 3. The dsRNA content in mRNA synthesized with CleaScript™ T7 RNA polymerase is lower than that in mRNA synthesized with the wild-type enzyme after cellulose treatment.
Shipping and Storage
The products are shipped with dry ice and can be stored at -15℃ ~ -25℃ for one year.