Vaccinia Capping Enzyme

Basic product informationSource: E. coli carrying vaccinia virus capping enzyme geneReaction conditions: 1x Capping Buffer (50 mM Tnis-HC1, pH 8.0; 5 mM KC1; 1 mMMgaly; 1 mM DTT). Incubation at 37°CStorage buffer: 20mM Tnis-HC1 pH8.0; 100mMNaC1; 1mM DTT; 0.1mMEDIA; 0.1% TritonX-100; 50% (wv) GlycerolProduct DescriptionThe mRNA obtained by in vitro transcription has not undergone a series of intracellular modifications, does not have the Cap structure and PolyA tail, is easily degraded, easily activates the immune response, cannot bind to the ribosome initiation protein, and cannot initiate protein translation. Therefore, in the industrialized mRNA production, It is necessary to use vaccinia virus capping enzyme to cap the IVT mRNA to obtain the Cap0 structure at the 5'end of the mRNA, and further use 2'-O-methyltransferase to convert Cap0 to Cap1. The cap structure introduced by enzymatic capping is completely consistent with the natural cap structure in eukaryotes, which fundamentally reduces the immunogenicity of exogenous mRNA while protecting it from degradation, improving translation efficiency, and increasing intracellular protein production. Enzymatic capping can achieve a maximum capping efficiency of 100%, while capping by chemically synthesized cap analog structures has relatively low capping efficiency, and the structure of cap analogs is different from the natural cap structure.The vaccinia virus capping enzyme adds the 7-methylguanosine cap 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. Enzymatic reaction to cap RNA is a simple and effective method, which can significantly improve the stability and translation ability of RNA used for in vitro transcription, transfection and microinjection. This enzyme is composed of two subunits (D1 and D12) (Figure 1a). The D1 subunit performs the functions of RNA triphosphatase and guanosine transferase, and the D12 subunit performs the functions of guanine methyltransferase. It is necessary to add a complete Cap0 structure m7Gppp5´N (Figure 1b).This product can be used for the capping reaction of RNA produced by T7 RNA Polymerase and GMP Grade (GMP-E121, Novoprotein) reactions. The capping reaction is completed within one hour, with an efficiency close to 100% and ensuring the correct direction.Recombinant vaccinia virus capping enzyme is expressed by large-scale fermentation in Escherichia coli. It is produced with medicinal-specific raw materials and strictly controlled host protein residues and nucleic acid residues. Product production and quality management procedures comply with GMP standards to ensure the production process and all raw materials Traceable.Figure 1. The structure and mechanism of vaccinia virus capping. a. Co-crystal structure of vaccinia virus capping enzyme, GTP (red) and 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 consists of a 7-Methylguanosine is composed 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 adjacent RNA strands. The 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 the body. 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.Quality requirements Project Standard Method Exterior Clear Liquid Visual Inspection Visible Foreign Body Compliance Chinese Pharmacopoeia 2020 Edition Fourth Part 1 Lamp Inspection Method (General Rule 0904) Ph Value 7.5-8.5 Chinese Pharmacopoeia 2020 Edition Part Iv Ph Determination Method (General Principle 0631) Active 9.8Ku/Ml-10.2Ku/Ml Capping Modification And Efficiency Determination Method Purity ≥95% Chinese Pharmacopoeia 2020 Edition Part Iv High Performance Liquid Chromatography (General Principle 0512) Endonuclease Residue 004-Dna Degradation Does Not Exceed 10% 10U Enzyme And 004-Dna. Incubate At 37℃ For 3H Exonuclease Residue 019 Hind Ⅲ Dna Degradation Does Not Exceed 10% 10U Enzyme And 019 Hind Ⅲ Dna. Incubate At 37℃ For 3H Rnase Residue Degradation Of 293-Rna Does Not Exceed 10% 10U Enzyme And 293-Rna, Incubate At 37℃ For 1H Bacterial Endotoxin Content ≤10 Eumg Chinese Pharmacopoeia 2020 Edition Fourth Gel Limit Test Method (General Rule 1143) Exogenous Dna Residue ≤100 Pg/Mg Fluorescence Quantitative Pcr Host Protein Residue ≤50 Ppm Chinese Pharmacopoeia 2020 Edition Part Iv Method For The Determination Of Bacterial Protein Residues (General Rule 3412) Mycoplasma Detection Feminine Mycoplasma Detection Kit Heavy Metal Residue ≤10Ppm Chinese Pharmacopoeia 2020 Edition Fourth Heavy Metal Inspection Method (General Principle 0821) Follow the following specifications for production1. ISO 9001:2015, certified facility.2. "GMP Appendix-Cell Therapy Products" State Drug Administration.3. "General Introduction to Human Gene Therapy-Chinese Pharmacopoeia 2020" National Pharmacopoeia Commission.4. USP Chapter <1043>, Ancillary Materials for Cell, Gene, and Tissue-Engineered Products are used as excipients in cell therapy, gene therapy and tissue engineering products.5. USP Chapter <92>, Growth Factors and Cytokines Used in Cell Therapy Manufacturing Cytokines and growth factors used in the production of cell therapy products.6. Ph. Eur. General Chapter 5.2.12, Raw Materials of Biological Origin for the Production of Cell-based and Gene Therapy Medicinal Products.Product UsageCapping of mRNA before translation in vivo or in vitro and labeling of 5'end of mRNA.ApplicationsThe intact mRNA expresses GFP protein in the cell, the capping enzyme is compared with the cap analogPrecautions1. The RNA used for the capping reaction should be purified and dissolved in nuclease-free water before use. The solution must not contain EDTA and salt.2. When configuring the reaction system, 0.5 µl of RNase inhibitor (GMP-E125, Novoprotein) can be added, and an equal volume of RNase-free Water can be removed at the same time.3. Heating the RNA before reacting with the capping enzyme can eliminate the 5´ secondary structure of the transcription product. If the structure of the 5´ end of the transcription product is complex, the heating time can be extended to 10 minutes.4. SAM is unstable at pH 7-8, 37°C and needs to be freshly configured before the reaction starts. The amount of SAM can be calculated in advance, and the aliquoted 32 mM stock solution can be diluted to 2 mM working solution before the reaction starts. To avoid degradation of SAM, this working solution needs to be stored on ice.5. If the 5´ end of the transcription product is known to have a complex structure, the reaction time can be extended to 60 minutes to increase the capping efficiency.6. When labeling the 5´ end, the GTP stock solution should be diluted to 1–3 times the mRNA concentration. Specifications and Purity: Pharmaceutical grade. Related Document: https://ald-pub-files.oss-cn-shanghai.aliyuncs.com/aladdinsci/pdp/sds/1/V406454-SCI_a7ead7deb444371b31b40b9bb75e0a2e.pdf.