Reliable method for high quality His-tagged and untagged E. coli phosphoribosyl phosphate synthase (Prs) purification
Beata Walter , Aneta Szulc , Monika Glinkowska
AbstractPrs (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. In Escherichia 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 of E. coli Prs 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 from prs-2 allele variant which was previously related to thermo-sensitive growth. His-PrsG9S protein purified here, exhibited comparable activity as previously observed in-vivo suggesting 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 assays in-vitro.
|Journal series||Protein Expression and Purification, ISSN 1046-5928, e-ISSN 1096-0279, (N/A 70 pkt)|
|Publication size in sheets||0.50|
|Keywords in English||Escherichia coli, phosphoribosyl phosphate synthase, Prs, PRPP, activity, temperature-sensitiveprs-2mutant|
|License||Journal (articles only); published final; ; with publication|
|Score||= 70.0, 22-06-2020, ArticleFromJournal|
|Publication indicators||: 2016 = 0.627; : 2018 = 1.291 (2) - 2018=1.368 (5)|
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