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$1,400.00
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SKU
ko51607-1-1
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100婵炴挾鎸?liquid plasmid(Instantly):price2rd
Description
The vaccinia capping enzyme together with substrates, is capable to add the 7-methylguanosine capping structure (m7Gppp, Cap0) to the 5? end of the RNA. In eukaryotes, this structure is closely related to the stability, transport, and translation of mRNA. Capping RNA with an enzymatic reaction is a simple and effective method that can significantly improve the stability and translation capabilities of RNA used for in vitro transcription, transfection, and micro-injection. The vaccinia virus capping enzyme consists of two subunits (D1 and D12) (Figure 1a), and possesses RNA triphosphatase, guanosine transferase, and guanine methyltransferase activities, and they are all necessary for adding a complete Cap0 structure m7Gppp5?N to mRNA (Figure 1b).
This product can be applied to capping reaction of RNA generated by the T7 RNA Polymerase (GMP-E121, Novoprotein). The capping reaction is completed within an hour and the efficiency is close to 100% with correct direction.
Vaccinia Capping Enzyme is GMP Grade produced in E. coli. Our manufacturing processes are strictly controlled to ensure the end products free from host protein or nucleic acid contaminations and other impurities following the Pharmaceutical Manufacturing Guidelines. We guarantee the manufacturing and quality control comply with GMP regulation for tracking each and every step of the manufacturing process, including raw material sourcing.
This product has completed the DMF record of FDA and passed the HALAL certification.
Fig 1. The structure and mechanism of vaccinia virus capping. a. Co-crystal structure of vaccinia virus capping enzyme and GTP (red), SAH (rose red) (PDB 4CKB). The enzyme consists of two subunits, D1 and D12, and has the functions of RNA triphosphatase (blue), guanosine transferase (orange) and guanine methyltransferase (beige). This figure is quoted from Ramanathan, A., Robb, GB, & Chan, SH (2016). mRNA capping: biological functions and applications. Nucleic Acids Res 44 (16), 7511–7526. b. The cap structure of mRNA is composed of a 7-Methylguanosine consists of a 5’-5’ triphosphate bridge connected to the 5’ nucleoside of the mRNA chain. The Cap-0 structure is formed by the sequential reaction of three enzymes between adjacent RNA strands. Further formation of cap-1 structure requires the participation of 2-O methyltransferase. This modification can reduce the cellular innate immune response caused by RNA in vivo. This figure is quoted from Decroly, E., Ferron, F., Lescar, J. et al. (2012). Conventional and unconventional mechanisms for capping viral mRNA. Nat Rev Microbiol 10, 51–65.
Fig 2. Vaccinia Capping Enzyme (GMP-M062) can co-work with 2’-O-Methyltransferase (GMP-M072). When using new cell lines or translation systems, it is recommended to compare the translation efficiency and immunogenicity of Cap 0 and Cap 1-mRNA to determine the optimal Cap structure
Quality Control
| Element | Standard |
| Appearance | clear and transparent solution |
| Identification | Positive |
| Visible Particles | Meet the specification |
| pH | 7.5-8.5 |
| Activity | 10KU/ml-10.5KU/ml |
| Purity | ≥95% |
| Protein Content | Meet the specification |
| Endonuclease Residues | The degradation of substrate was ≤10% |
| Exonuclease Residues | The degradation of substrate was ≤10% |
| RNase Residues | The degradation of substrate was ≤10% |
| Bacterial Endotoxins | ＜5EU/ml |
| Exogenous DNA Residues | ≤100pg/mg |
| Host-cell Protein Residues | ≤50ppm |
| Mycoplasma | Negative |
| Heavy Metals | ≤10ppm |
| Microbial Limit | Total aerobic microbial count≤1cfu/10ml, total yeasts and molds count≤1cfu/10ml |
Product Details
| Item | Details |
| Storage | At -20±5℃. |
Components
| SKU | Components | Volume |
| GMP-M062-01A | Vaccinia Capping Enzyme, GMP Grade (10U/μl) | 50μl |
| GMP-M062-M001 | Vaccinia Capping Enzyme, GMP Grade (10U/μl) | 1ml |
| GMP-M062-M010 | Vaccinia Capping Enzyme, GMP Grade (10U/μl) | 10ml |
| GMP-M062-M050 | Vaccinia Capping Enzyme, GMP Grade (10U/μl) | 50ml |
Notes
SAM is unstable at pH 7–8, 37 °C and needs to be freshly prepared before the reaction starts. The amount of SAM can be calculated in advance, and the aliquoted 32 mM stock solution is diluted into a 2 mM working solution before the reaction starts. To avoid SAM degradation, the working solution needs to be kept on ice.
Before starting reaction, the RNA applied for capping should be purified and dissolved in nuclease-free water. The solution for use must NOT contain any EDTA or salt.
RNA transcript with 5’ end secondary structure can be an obstacle for capping. Heating the RNA sample can remove complex structure of the transcript. We may extend heating time to 10 minutes and extend the capping reaction time to 60 minutes.
Capping from Cap0 to Cap1 naturally exists in eukaryotic cells, which enhance translation efficiency.
Vaccinia Capping Enzyme (GMP-M062) can co-work with 2’-O-Methyltransferase (GMP-M072). When using new cell lines or translation systems, it is recommended to compare the translation efficiency and immunogenicity of Cap 0 and Cap 1-mRNA to determine the optimal Cap structure.
When adding 0.5 ?l of RNase inhibitor (GMP-E125, Novoprotein) to the reaction system (shown below in “Manual” part), please deduct equal volume of RNase-free water.
When used for RNA 5? -end labeling, GTP should be diluted to 1-3 times of the mol concentration of mRNA.
When used for the capped RNA 3’-poly (A) tailing, we may apply E. coli Poly(A) Polymerase, GMP Grade (GMP-M012, Novoprotein).
The sample needs to be purified before transfection.
Do NOT store the product at -70°C or an even lower temperature.
In case of white precipitation in reaction buffer (shown below in “Manual” part), incubate it at 37 ° C for 5 mins, blend it, totally dissolving precipitation.
Related Products
| Cat. No. | Product Name | Cat. No. | Product Name |
| GMP-M072 | mRNA Cap 2?-O-Methyltransferase, GMP Grade | GMP-E125 | RNase Inhibitor, GMP Grade |
| GMP-M036 | Pyrophosphatase, Inorganic (yeast), GMP Grade | GMP-E127 | DNase I, GMP Grade |
| GMP-M012 | E. coli Poly(A) Polymerase, GMP Grade | GMP-E121 | T7 RNA Polymerase, GMP Grade |
| GMP-EB62 | 10× Capping Reaction Buffer, GMP Grade | GMP-S062 | SAM, GMP Grade |
| GMP-S024N | GTP, GMP Grade |
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