Engineered Baculovirus carrying the Thermostable Pyrophosphatase gene from S. acidocaldarius inoculated in Trichoplusia ni.
Thermostable Inorganic Pyrophosphatase
For research only
Our Thermostable Inorganic Pyrophosphatase is a vital enzyme for nucleic acid synthesis, improving the efficiency of molecular biology workflows. It catalyzes the hydrolysis of inorganic pyrophosphate (PPi) into orthophosphate, driving reactions forward, and is highly robust, maintaining stability even at elevated temperatures.
Product specification
Source
Unit Definition
One unit (U) will release 1.0 µmol of inorganic orthophosphate per minute at pH 9.0 at 75°C.
Storage Buffer
10 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM DTT, 0.1 mM EDTA and 50% Glycerol.
Molecular Weight
20.2 kDa.
Format
Liquid.
Downloads
Quality Control Analysis
Enzyme Activity
Determined by Absorption Spectrophotometry: The phosphate released after pyrophosphate hydrolysis by pyrophosphatase is measured by means of a color reaction. Dilutions of the enzyme in 50mM Tris-HCl pH 9.0, 10mM MgCl2 and 10mM sodium pyrophosphate are assayed.
After a 10 min incubation at 75°C, the 2-orthophosphate product reacts with ammonium molybdate to form phosphomolybdic acid. The phosphomolybdic acid is reduced by ferrous sulphate giving a blue colour to the reaction. This colour can be measured at 660 nm. A phosphate standard curve is used to quantify the product formed.
Protein Purity
Evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by Coomassie stain. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the protein of interest band in the diluted sample.
DNase Contamination
Determined by Fluorometric Assay. Use of a fluorogenic substrate that emits fluorescence after cleavage by the presence of DNases.
Residual (genomic) DNA Contamination
Evaluated by quantitative PCR: Amplification of specific genomic sequence of T ni.
Applications
RNA synthesis
Hydrolysis of PPi enhances in vitro transcription, increasing RNA yield and accelerating reaction rates.
DNA synthesis
Removal of PPi prevents polymerase inhibition, improving yields in isothermal amplification and long DNA synthesis.
References
1. Cunningham, P.R. and Ofengand, J., Use of inorganic pyrophosphatase to improve the yield of in vitro transcription reactions catalyzed by T7 RNA polymerase, Biotechniques, 9, 713-714, 1990.
2. Tabor, S., Richardson, C.C., DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Effect of pyrophosphorolysis and metal ions, J. Biol. Chem., 265, 8322-8328, 1990.
3. Dean, B.F., et al., Rapid amplification of plasmid and phage DNA using phi29 DNA polymerase and multiply-primed Rolling Circle amplification, Genome Res., 11, 1095-1099, 2001.
A case study in reducing enzyme costs
Explore how we produced Thermostable Inorganic Pyrophosphatase resulting in >70% cost savings for our client, a leading pharmaceutical company in the mRNA vaccine production.
Request a sample or discuss quantities
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