mRNA Cap 2´-O-Methyltransferase

Basic product informationSource: E. coli strain carrying the 2'-O-methyltransferase gene of the vaccinia virus mRNA cap structure.Reaction conditions: 1× Capping Reaction Buffer (50 mM Tnis-HC1, pH 8.0; 5 mMKCl; 1 mMMgCl; 1 mM DTT), incubated at 37CStorage buffer: 20mM Tnis-HCl pH8.0; 100mMNaC1; 1mMDTT; 0.1mM EDTA; 0.1% TritonX-100; 50% (vv) 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.mRNA Cap 2´-O-Methyltransferas can use S-adenosylmethionine (SAM) as a methyl donor, at the 2´- end of the RNA 5´ end next to the first nucleotide of the cap structure (Cap 0) Add a methyl group to O to form mRNA with Cap 1 structure. The Cap1 structure can enhance the translation efficiency of mRNA, thereby increasing the expression level of the protein encoded by the mRNA after transfection. This enzyme can only use RNA with a 7-methylguanosine cap structure (m7GpppN) as a substrate, and will not act on RNA with pN, ppN, pppN or GpppN at the 5´ end. RNA with 7-methylguanosine cap structure (m7GpppN) can be prepared by Vaccinia Capping System (Cat. No: GMP-M062, Novoprotein).This product is expressed in large-scale fermentation using Escherichia coli, is produced with medicinal specifications of raw materials, and strictly controls host protein residues, nucleic acid residues, etc., and conforms to GMP standard product production and quality management procedures to ensure that the production process and all raw materials can be traced.Figure 1. mRNA cap structure 2´ -O-methyltransferase mechanism of action. The cap structure of mRNA consists of a 7-methylguanosine connected to the 5'nucleoside of the mRNA chain via a 5'-5' triphosphate bridge. 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, which 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, 51-65Quality requirement 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 49Kuml-51Ku/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% 50U Enzyme And 004-Dna. Incubate At 37℃ For 3H Exonuclease Residue 019 The Degradation Of Hinditi Dna Does Not Exceed 10% 50U Enzyme And 019 Hindiii Dna. Incubate At 37℃ For 3H Rnase Residue Degradation Of 293-Rna Does Not Exceed 10% 50U Enzyme And 293-Rna. Incubate At 37℃ For 1H Endotoxin Content Of Aspergillus ≤10 Eu/Mg 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 ≤10 Ppm Chinese Pharmacopoeia 2020 Edition Fourth Vehicle 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 UsageThe mRNA of Cap0 structure is 2´-O methylated (Cap1) to improve the efficiency of mRNA translation and expression.ApplicationsThe intact mRNA expresses GFP protein in the cell, the capping enzyme is compared with the cap analogPrecautions1. The RNA used in the reaction should be purified and dissolved in RNase-free water before use. The solution must not contain EDTA and salt;2. When configuring the reaction system, it is recommended to use an RNase inhibitor to enhance the stability of RNA in the reaction. 0.5µl of RNase inhibitor (Cat. No: GMP-E125, Novoprotein) can be added, and the same volume of RNase-free water can be removed at the same time;3. Heating RNA before the reaction can remove the secondary structure at the 5´ end. 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 32 mM stock solution can be diluted to 4 mM working solution before the reaction starts. To avoid degradation of SAM, this working solution needs to be stored on ice. Specifications and Purity: Pharmaceutical grade.