Inorganic pyrophosphates are inevitably produced in the process of mRNA transcription in vitro. These substances have a great inhibitory effect on transcription. Inorganic pyrophosphatase (PPase) can hydrolyze the inorganic pyrophosphates produced in nucleic acid amplification experiments, promote the shift of reaction equilibrium to the product generation end, and increase the amount of products.
The molecular weight of PPase (pyrophosphatase, inorganic, inorganic pyrophosphatase) is about 63kd, which can catalyze the hydrolysis of inorganic pyrophosphate to produce orthophosphate: P2O74_+H2O+PPase→2HPO42_. In the nucleic acid amplification experiment, PPase can hydrolyze the inorganic pyrophosphate generated with the reaction to avoid its inhibition on the reaction system. The removal of pyrophosphate can shift the reaction equilibrium to the product generation end.
This product is a GMP level recombinant inorganic pyrophosphatase (yeast source) expressed by large-scale fermentation of E. coli. It is produced with raw and auxiliary materials of medicinal specifications, and the host protein residue and nucleic acid residue are strictly controlled. The product production and quality management procedures in line with GMP specifications ensure that the production process and all raw and auxiliary materials can be traced.
Quality requirements
| project |
standard |
| appearance |
Clear liquid |
| Visible foreign matter |
Compliance with regulations |
| PH value |
7.5±8.5 |
| activity |
98U/ml-102U/ml |
| purity |
≥95% |
| Endonuclease residues |
Degradation of substrate shall not exceed 10% |
| Exonuclease residues |
Degradation of substrate shall not exceed 10% |
| RNase residue |
Degradation of substrate shall not exceed 10% |
| Bacterial endotoxin content |
≤10EU/ml |
| Exogenous DNA residue |
≤100pg/mg |
| Host protein residue |
≤50ppm |
| Mycoplasma detection |
negative |
| Heavy metal residues |
≤10ppm |
|
Follow the following specifications
1. 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 Committee.
4. USP chapter <1043>, adjuvant materials for cell, gene, and tissue engineered products.
5. USP chapter <92>, growth factors and cytokines used in cell therapy manufacturing.
6. Ph. Eur. General chapter 5.2.12, raw materials of biological origin for the production of cell-based and gene therapy medical products.
Product features
1. hydrolyze inorganic pyrophosphate.
2. DNA synthesis: significantly enhance DNA replication ability.
3. RNA synthesis: increase RNA production in in vitro transcription reaction.
4. The optimal reaction temperature is 25℃, and the enzyme can be inactivated at 65℃ for 10min.
Product usage
1. optimize RNA transcription: improve the RNA yield of in vitro transcription reaction.
2. remove PPI contamination from reagents for SNP genotyping by pyrophosphate assay.
3. promote the synthesis of protein, RNA and DNA.
4. catalyze the reaction of PPI + H2O → 2pi.
5. ssr-pcr optimization：Improve efficiency and increase DNA production.
Activity definition
Catalytic inorganic pyrophosphate formation 1 per minute under standard reaction conditions μ The amount of enzyme required for mol phosphate was defined as 1 active unit.
Preservation system
20 mM Tris-HCl; 100 mM NaCl; 1 mM DTT; 0.1 mM EDTA; 50% (v/v) Glycerol; pH 8.0。
Storage temperature
-20±5 ℃。
Matters needing attention
1. the enzyme has activity in various reaction buffers. Generally, the enzyme can be directly added in HDA, lamp and other experiments.
2. the dosage of the enzyme needs to be optimized in different experiments, usually adjusted at the concentration of 0.05~1u/ml.
3. the optimum reaction temperature of the enzyme was 25 ℃, and it was active at 16~37 ℃, and the enzyme could be inactivated at 65 ℃ for 10min.
4. cofactor: mg2+ is necessary for enzyme activity.
B2320504