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Reliable method for high quality His-tagged and untagged E. coli phosphoribosyl phosphate synthase (Prs) purification
- Publication Year :
- 2019
- Publisher :
- Cold Spring Harbor Laboratory, 2019.
-
Abstract
- Prs (phosphoribosyl pyrophosphate synthase) is a broadly conserved protein that synthesises 5-phosphoribosyl 1-pyrophospate (PRPP); a substrate for biosynthesis of at least 10 enzymatic pathways including biosynthesis of DNA building blocks – purines and pyrimidines. InEscherichia coli, it is a protein of homo-hexameric quaternary structure, which can be challenging to work with, due to frequent aggregation and activity loss. Several studies showed brief purification protocols for various bacterial PRPP synthases, in most cases involving ammonium sulfate precipitation.Here, we provide a protocol for expression ofE. coliPrs protein in Rosetta (DE3) and BL21 (DE3) pLysE strains and a detailed method for His-Prs and untagged Prs purification on nickel affinity chromatography columns. This protocol allows purification of proteins with high yield, purity and activity. We report here N-terminally His-tagged protein fusions, stable and active, providing that the temperature around 20 °C is maintained at all stages, including centrifugation. Moreover, we successfully applied this method to purify two enzyme variants with K194A and G9S alterations. The K194A mutation in conserved lysine residue results in protein variant unable to synthetize PRPP, while the G9S alteration originates fromprs-2allele variant which was previously related to thermo-sensitive growth. His-PrsG9S protein purified here, exhibited comparable activity as previously observedin-vivosuggesting the proteins purified with our protocol resemble their physiological state.The protocol for Prs purification showed here indicates guidance to improve stability and quality of the protein and to ensure more reliable results in further assaysin-vitro.
- Subjects :
- 0106 biological sciences
Recombinant Fusion Proteins
Phosphoribosyl Pyrophosphate
medicine.disease_cause
01 natural sciences
Chromatography, Affinity
03 medical and health sciences
chemistry.chemical_compound
Affinity chromatography
Biosynthesis
010608 biotechnology
Escherichia coli
medicine
Cloning, Molecular
Ammonium sulfate precipitation
030304 developmental biology
chemistry.chemical_classification
0303 health sciences
ATP synthase
biology
Phosphoribosyl pyrophosphate
Temperature
Enzyme
chemistry
Biochemistry
biology.protein
Protein quaternary structure
Biotechnology
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Accession number :
- edsair.doi.dedup.....b2523f74d1fa149ea989df73d936d194
- Full Text :
- https://doi.org/10.1101/864173