mRNA Vaccinia Capping Enzyme GMP-grade

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Cat Number

PIPB-0795

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Product Detail Description Scientific Support Customer Q&A

Storage

Store at -20 ℃

Appearance

Clear and transparent liquid

Standard

Facility GMP

Qualification

DMF

General Description

Vaccinia Virus Capping Enzyme is a highly effective, recombinant enzyme manufactured to cGMP standards to ensure regulatory compliance for use in the production of therapeutic mRNA and vaccines. It is supplied as a liquid, clarified and purified from a recombinant source and tested for freedom from endonucleases, exonucleases and RNases.

Mechanism of Action

Vaccinia Virus Capping Enzyme is a heterodimer consisting of D1 and D12 subunits that display the various enzymatic activities involved in cap synthesis. D1 and D12 function cooperatively to first catalyze sequential formation of the 5'-end, and then the subsequent methylation reactions to install the correct 7-methylguanosine cap structure at the RNA terminus.

Application

The most widely used application of Vaccinia Virus Capping Enzyme is for in vitro capping of mRNA, for high fidelity RNA modification. It is essential for the large scale manufacture of mRNA therapeutics and vaccines, where successful translation is critical. It is also used in research applications for preparing mRNA for in vitro translation assays and for site-specific 5'-end labeling of mRNA transcripts for a variety of molecular studies.

Crystal structure of the full-length vaccinia virus mRNA capping enzyme (D1/D12 heterodimer) in the context of three catalytic modules (RNA triphosphatase or TPase, guanylyltransferase or GTase, and guanine-N7-methyltransferase or MTase) used sequentially for caps synthesis.
The TPase module is a triphosphate-tunnel metalloenzyme with a basic roof and an acidic floor to coordinate the γ-phosphate of the nascent RNA through Lys161 and Glu126, respectively. The GTase module has a closed NTase–OB-fold conformation that is locked by an extensive 2155 Å² TPase–GTase interface. Disruption of this interface by mutations of interfacial residues L47A-L50A-T51A or K478A results in the loss of GTase activity without affecting TPase activity, indicating the allosteric coupling of the two modules. GTP is bound in the GTase active site in a suboptimal geometry, and must be repositioned upon Mg2+ binding for Lys260 nucleophilic attack. The MTase domain is connected to the other modules through a flexible linker and is primed for activity by D12, a degenerate 2′-O-methyltransferase homolog that locks the AdoMet-binding helix αK in place.

Fig. 1 Crystal Structure of Vaccinia Virus mRNA Capping Enzyme. (Kyrieleis O J P.; et al. 2014)

References

Kyrieleis O J P, et al. Crystal structure of vaccinia virus mRNA capping enzyme provides insights into the mechanism and evolution of the capping apparatus. Structure, 2014, 22(3): 452-465.