E. coli Pyrophosphatase

• Catalyzes the hydrolysis of inorganic pyrophosphate (PPi) to form orthophosphate
• Removal of pyrophosphate (PPi) and its inhibitory effects enhances RNA and DNA synthesis reactions

E. coli Pyrophosphatase catalyzes the Mg-dependent reaction of P₂O₇^-4 + H₂O → 2HPO₄^-2.

This enzyme is supplied in 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA and 50% glycerol.

• Storage temperature: –25°C to –15°C
• Molecular weight: 19,704 Daltons

The recombinant enzyme protein is produced by a recombinant E. coli strain carrying the E. coli Pyrophosphatase gene. One unit is the amount of enzyme that will liberate 1 µmol of phosphate per minute from inorganic pyrophosphate at 37°C and pH 8.5.

Usage Instructions

The E. coli Pyrophosphatase is widely used for in vitro transcription (IVT) or RNA synthesis reactions to reduce inhibitory effects of PPi. As a starting point for in vitro RNA synthesis reaction, add 0.1-1 units per mL of E. coli Pyrophosphatase to identify the optimal concentration.

Notes:  

E. coli Pyrophosphatase catalysis is Mg²⁺-dependent, therefore, it is important to have Mg²⁺ in the reaction buffer.

References: 

1. Lahti, R. et al. (1988) J. Bacteriol., 170(12), 5901-7.

2. Baykov, A.A. et al. (1996) Biochemistry, 35(15), 4655-61.

3. Taussky, H.H. and Shorr, E. (1953) J. Biol. Chem., 202(2), 675-85.

Quality Control

Enzyme dilutions are added to 30 mM Tris HCl (pH 8.5), 1.5 mM MgCl₂ and 1.5 mM sodium pyrophosphate. After a 10-minute incubation at 37° C, the product formed, 2- orthophosphate, is reacted with ammonium molybdate to form phosphomolybdic acid. The phosphomolybdic acid is then reduced by ferrous sulfate under weak acidic conditions to create a blue color, the absorbance of which is measured at 660 nm. The product formed is extrapolated from a standard phosphate curve generated from the ammonium molybdate/ferrous sulfate reaction. The assay is based on that described by Taussky and Shorr (3). 

Protein concentration is determined by OD₂₈₀ absorbance. 

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the band's mass corresponding to the protein of interest in the diluted sample. 

Single-stranded exonuclease is determined in a 50 µL reaction containing a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C. 

Double-stranded exonuclease activity is determined in a 50 µL reaction containing a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C. 

Double-stranded endonuclease activity is determined in a 50 µL reaction containing 0.5 µg of plasmid DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C. 

E. coli contamination is evaluated using 5 µL replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus. 

• DNA and RNA synthesis and modification