CM4010
Carbomenu has protein expression platform in both prokaryotic cells (E.coil) and eukaryotic cells (Yeast/CHO-k1/293). Carbomenu is able to deal with kilogram level customized protein production orders. Our product line can be optimized according to customer orders to meet personalized needs.
Information
Known as: | Capping Enzyme |
Species： | Vaccinia |
Cat.No.: | CM4010 |
Expression Host： | Ecoli. |
Package: | Customed |
Price: | Inquiry |
Introduction
Vaccinia capping enzyme is composed of two subunits (D1 and D12). The D1 subunit carries three enzymatic activities RNA including triphosphatase, guanylyltransferase and guanine methyltransferase, which are all necessary for addition of a complete Cap 0 structure, m7Gppp5'N to 5' triphosphate RNA.
In vitro transcripts can be capped in less than one hour in the presence of the capping enzyme, reaction buffer, GTP, and the methyl donor, SAM. Capping is nearly 100% efficient and all capped structures are added in the proper orientation, unlike co-transcriptional addition of some cap analogs.
Storage
Transportation with Ice pack.
Stored at -20℃, stable for one year. It is recommended to subpackage to avoid repeated freeze-thaw.
Enzyme activity
1 unit (U) is defined as the amount of enzyme required for incorporation of 10pmol (α -32p) GTP into an 80 nucleotide (80nt) transcription product within one hour at 37℃.
Reaction condition
1xCapping Buffer(50mM Tris-HCl, pH 8.0; 5mM KCl;1mM MgCl2; 1mM DTT), incubated at 30℃.
Storage Buffer
20mM Tris-HCl pH8.0; 100mM NaCl; 1mM DTT; 0.1mM EDTA; 0.1% Triton-X; 50%(v/v)Glycerol.
Notes
1. RNA should be purified and dissolved in Rnase-free water before use. Solution should not contain any EDTA and salt ions.
2. When configuring the reaction system, 0.5 µ L RNase inhibitor (GMP-E125, Novoprotein) can be added, in the meantime equal volume of Rnase-free Water should be removed.
3.Before reaction, it is recommended to heat at 65℃ for 5mins to remove the secondary structure of RNA. If the 5´terminal structure of the transcript is complex, the time can be extended to 10mins.
4. SAM is poor stability at pH 7-8 and 37°C, so it needs to be prepared before use. The amount of SAM required can be calculated in advance. Before reaction the stock solution (32mM) can be diluted into 4mM working solution. To prevent SAM degradation, the working fluid should be stored on ice.
5. If the 5´ terminal structure of the transcription product is known to be complex, the reaction time can be extended to 60 minutes to improve the capping efficiency.
6. When labeling the 5´ end, the GTP stock solution should be diluted to 1-3 times the concentration of mRNA.
FOR RESEARCH USE ONLY
References
1.Conventional and unconventional mechanisms for capping viral mRNA.Etienne Decroly, François Ferron, Julien Lescar & Bruno Canard. Nature Reviews Microbiology.2012, pages51–65
2.Crystal structure of vaccinia virus mRNA capping enzyme provides insights into the mechanism and evolution of the capping apparatus.Otto J.P. Kyrieleis,Jonathan Chang,Marcos de la Peña,Stewart Shuman,and Stephen Cusack1. Structure. 2014 Mar 4; 22(3): 452–465.
3. Characterization of a trifunctional mimivirus mRNA capping enzyme and crystal structure of the RNA triphosphatase domain. Benarroch D, Smith P, Shuman S. Structure. 2008;16:501–512.
