Modification of histidine repeat proteins by inorganic polyphosphate

Protein purification via consecutive histidine–polyphosphate interaction

“…We performed a binding assay of the polyP resin with human protein MafB (a transcription factor) and yeast protein Snf1 (AMP‐activated Ser/Thr protein kinase), both of which contain internal and consecutive histidines in their wild‐type sequences. Notably, proteins expressed from both constructs capable of binding to polyP in NuPAGE analysis do not have a 6xHis‐tag at any terminus (Neville et al, 2023 ). Prior to polyP binding experiment, they were fused with an MBP‐tag, then purified through amylose resin and size exclusion chromatography (SEC).…”

“…Our lab recently made a serendipitous discovery related to polyphosphate (polyP, Figure S1 ) binding to proteins containing histidine‐repeat via non‐covalent interaction (Neville et al, 2023 ). PolyP, a linear polymer with inorganic phosphate residues, is found in all domains of life, ranging from bacterial to human cells (Kornberg et al, 1999 ), robustly binds to histidine‐repeat or histidine‐rich proteins (Malik et al, 2021 ), detectable through mobility shift on NuPAGE gel (Azevedo et al, 2015 ; Bentley‐DeSousa et al, 2018 ).…”

Protocol for detecting histidine polyphosphate modification of human proteins via MBP-tagged expression in E. coli

“…However, we have also used varying polyP chain-lengths and concentrations, see Figures 1E and 1F in Neville et al., (2023) for details on the range of polyP-dependent electrophoretic shifts. 1 PolyP preparation. Prepare a 100 mM stock solution of polyP 700 (Kerafast, Cat#EUI003).…”

“… Note: pH can be altered by varying the ratio of MOPS and Tris to avoid confounding ionic strength, which might otherwise occur if pH were adjusted using concentrated NaOH or HCl. 1 Compare relative elution volumes using relative absorbance values and by running fractions on a NuPAGE gel. …”

“… 1 N/A N/A Recombinant DNA Plasmids: see Table S3 (Neville et al., 2023). 1 N/A N/A Other BamHI-HF New England Biolabs Cat#R3136L XhoI New England Biolabs Cat#R0146S GeneJET Purification Kit Thermo Fisher Scientific Cat#K0701 T4 DNA ligase New England Biolabs Cat#M0202L Lysogeny broth BioShop Cat#LBL407.5 GeneJET Plasmid Miniprep Kit Thermo Fisher Scientific Cat#K0502 IPTG BioShop Cat#IPT001 Dpn1 New England Biolabs Cat #R0176L Terrific broth BioShop Cat#TER409.5 Baffled Erlenmeyer flask MilliporeSigma Cat#CLS44501L Lysozyme BioShop Cat#LYS702 Amylose resin New England Biolabs Cat#E8021L Empty chromatography columns Bio-Rad Cat#7321010 Centrifugal filter MilliporeSigma Cat#UFC9050 Superdex 200 Increase 10/300 GL Cytiva Cat#28990944 Immoblion-FL PVDF membranes MilliporeSigma Cat#IPFL00010 Superdex 200 16/600 Cytiva Cat#28989335 Trans-Blot Turbo transfer system Bio-Rad Cat#1704150 Ponceau S staining solution Thermo Fisher Scientific Cat#A40000278 …”