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

Summary

Polyphosphate exhibits a unique post-translational modification-like function, known as histidine polyphosphate modification (HPM), marked by a robust non-covalent interaction with histidine repeat proteins. Here, we present a protocol for detecting HPM of human proteins via maltose-binding protein-tagged expression in E. coli. We describe steps for detecting HPM by observing electrophoretic mobility shifts on NuPAGE gels followed by western blot. We then detail procedures for analyzing the influence of ionic strength and pH on HPM.

For complete details on the use and execution of this protocol, please refer to Neville et al.¹

Graphical abstract

Highlights

• •Expression of HRPs using MBP-tagged pET-based vector (HT31)
• •Identifying histidine-polyP modification targets with electrophoretic NuPAGE shifts
• •Testing polyP-binding conditions with immobilized HRP polyP pull-down assays and SEC

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Polyphosphate exhibits a unique post-translational modification-like function, known as histidine polyphosphate modification (HPM), marked by a robust non-covalent interaction with histidine repeat proteins. Here, we present a protocol for detecting HPM of human proteins via maltose-binding protein-tagged expression in E. coli. We describe steps for detecting HPM by observing electrophoretic mobility shifts on NuPAGE gels followed by western blot. We then detail procedures for analyzing the influence of ionic strength and pH on HPM.

Before you begin

This protocol outlines the procedure to screen for histidine polyphosphate modification (HPM) targets using recombinantly expressed proteins from E. coli BL21(DE3) RIPL cells. However, HPM hits have also been successfully screened for in Saccharomyces cerevisiae cells using a GFP-tagged library. Here we describe polyP-protein target identification using NuPAGE analysis and biochemical characterization of this modification using size exclusion chromatography (SEC) and pull-down assays to examine the effects of pH and ionic strength on HPM. This protocol has been used for various HPM targets, including MafB, a human transcription factor (UniProt Q9Y5Q3). Herein we outline cloning details (Figures 1A and 2B) and HPM detection using NuPAGE analysis (Figure 1B), as well as SEC (Figure 2A) using wild type MafB and a histidine deletion mutant.

Figure 1.

NuPAGE detection of HPM using MBP-tagged protein via the HT31 vector

(A) Map of a pET16-based vector called HT31 containing a MafB histidine deletion sequence within its multiple cloning site (N-MBP-TEV-MafB ΔHis-C). E. coli cells containing HT31 will gain ampicillin resistance and protein expression is inducible by IPTG. TEV protease recognition and cleavage site is positioned C-terminal of the MBP tag for downstream MBP tag cleavage (schematic created in SnapGene).

(B) NuPAGE analysis of a wild type histidine repeat protein (WT MBP-MafB) with a histidine deletion mutant (MBP-MafB ΔHis) to confirm that polyP binding is histidine dependent. HPM was compared to the lysine polyphosphorylation shift of MBP-Top1 (52–125), which contains a polyacidic and lysine rich (PASK) motif.² MBP was used as a negative shift control. Approximate molecular weights of MBP, WT MBP-MafB, MBP-MafB ΔHis and Top1 in the absence of polyP are 41, 78, 77 and 51 kDa, respectively. Lower, non-specific molecular weights are due to protein degradation and impurities from purification.

Figure 2.

Using SEC to demonstrate that MafB polyP modification is histidine dependent in solution

(A) SEC of MBP-MafB (80–232) fusion incubated with 5 mM polyP₇₀₀ or 5 mM monomeric NaH₂PO₄ in no-salt buffer (NuPAGE mimetic). Note that the full-length MBP-MafB fusion aggregates and elutes in the void volume, thus, the truncated fusion is used for SEC analysis. Elution peaks at 8.5 mL for MBP-MafB ΔHis no polyP and MBP-MafB ΔHis with polyP samples are due to a high molecular weight contaminant.

(B and C) Schematic of full-length MBP-MafB (1–323) and the MBP-MafB fusion truncation (80–232) containing the histidine rich region (131–167) which is removed in the ΔHis samples.

Cloning of histidine repeat proteins

Timing: 1 day

• 1.PCR amplification of the desired histidine rich protein (HRP) gene from cDNA template with primers that add restriction enzyme cut sites (see multiple cloning site in Figure 1A).

Alternative: Commercially synthesized DNA encoding the desired HRP can be ordered. This has the advantage of codon optimization for protein overexpression in E. coli.

• 2.
  Clone HRP into a pET-based vector which includes an N-terminal MBP tag for enhanced solubility and a tobacco etch virus (TEV) protease cleavage site for downstream usage (see troubleshooting, problem 2).

  Note: We use and recommend a custom pET16-based vector called HT31 (N-MBP-TEV-insert-C), which is available upon request from the authors (Figure 1A).

  • a.
    Digest HT31 vector using restriction enzymes corresponding to the cut sites added to the desired insert.

    Note: For WT MBP-MafB (Figure 1) we use BamHI and XhoI (NEB, Cat#R3136L and Cat#R0146S).

  • b.
    Purify PCR DNA fragments using a GeneJET Purification Kit according to manufacturer instructions (Thermo Fisher Scientific, Cat#K0701).
  • c.
    Ligate DNA fragments into HT31 vector using T4 DNA ligase (NEB, Cat#M0202L).
• 3.
  Transform the MBP-tagged HRP plasmid into E. coli DH5α cells (Agilent, Cat#200231):

  • a.
    Add 40 ng of plasmid DNA to 100 μL of competent E. coli DH5α cells. Mix well and rest on ice for 25 min before heat shocking for 1 min on 40°C heat block and returning to ice for 5 min.
  • b.
    Add 500 μL of lysogeny broth (LB) (BioShop, Cat#LBL407.5) to cell mixture and mix well. Incubate at 37°C in a shaker at 100 g for 45 min before plating 100 μL of cell mixture onto an LB agar plate containing 100 μg mL⁻¹ ampicillin (for HT31).
  • c.
    Confirm positive transformants with colony PCR and whole plasmid sequencing following plasmid miniprep purification (Thermo Fisher Scientific, Cat#K0502).

Note: Make a frozen stock of successful transformants in E. coli DH5α cells grown for 18 h in LB and supplemented with 10% DMSO (BioShop, Cat# DMS555) and stored at ‒80°C.

• 4.For protein expression, repeat the transformation protocol with the purified sequenced plasmid using BL21(DE3) RIPL E. coli cells (Agilent, Cat#230280).

Note: These cells allow for inducible protein expression via isopropyl β-D-1-thiogalactopyranoside (IPTG) (BioShop, Cat#IPT001). RIPL denotes extra tRNAs in this strain to improve codon bias issues.

Deletion or mutagenesis of histidine repeat regions

Timing: 1 day (not including transformation or sequencing)

• 5.Select proteins that confer polyP-dependent electrophoretic shifts on NuPAGE gels (see NuPAGE analysis section) for mutagenesis or deletion studies to confirm histidine-dependent shifting.
• 6.
  Perform PCR based site directed mutagenesis:

  • a.
    Design custom mutagenic primers made to flank the region to be deleted or mutated. This results in a linear plasmid DNA product. For more details see Watson et al., (2019).³
  • b.
    Remove plasmid template DNA by Dpn1 restriction enzyme digestion (NEB, Cat#R0176L).
• 7.
  Transform directly into E. coli DH5α cells:

  • a.
    Follow transformation protocol outlined in step 3 of cloning HRPs.
  • b.
    Verify mutagenesis by sequencing.

Small-scale expression of HRPs

Timing: 2 days (not including NuPAGE analysis)

• 8.
  Using a HRP BL21(DE3) RIPL cell transformant, inoculate 5 mL of LB supplemented with selective antibiotic (100 μg mL⁻¹ ampicillin for HT31).

  • a.
    Grow culture for 18 h at 37°C in a shaker at 100 g.
  • b.
    Sub-culture: Transfer 50 μL of overnight culture into 5 mL of fresh LB media (1/100 dilution) with selective antibiotic. Grow at 37°C until OD₆₀₀ = 0.6 (∼3 h).
• 9.
  Induce with IPTG:

  • a.
    Allow culture to cool down to 20°C before adding IPTG (1 mM final concentration) to culture.
  • b.
    Grow for 18 h at 20°C in a shaker at 100 g.
• 10.
  Collect and lyse cell pellet:

  • a.
    Spin the entire 5 mL culture at 4550 g for 10 min at 4°C to pellet the cells.
  • b.
    Remove supernatant and resuspend cell pellet in a 1:1 ratio (40 μL final volume) of lysis buffer and 2X Laemmli Buffer (120 mM Tris pH 6.8, 100 mM DTT, 20% glycerol, 4% SDS, 0.2% bromophenol blue) (BioShop, Cat#TRS001, Cat#DTT001, Cat#GLY004, Cat#SDS999, and Cat#BRO222). Vortex briefly. Boil at 95°C for 5 min.

Pause point: Cell pellets or lysates in buffer can be flash frozen in liquid nitrogen and stored at ‒80°C for several months.

Large-scale expression and purification

Timing: 3 days (not including NuPAGE analysis)

• 11.Repeat steps 7‒10a using a larger initial culture volume of 50 mL and final volume of 500 mL Terrific Broth (BioShop, Cat#TER409.5) supplemented with ampicillin for HT31 (100 μg mL⁻¹).

Note: Proper aeration during protein expression is important for cell growth. Using a 1 L baffled Erlenmeyer flask (Millipore Sigma, Cat#CLS44501L) at half capacity tends to increase oxygenation. As well, we suggest using nutrient-dense Terrific Broth instead of LB for large-scale expressions to promote higher plasmid yields and thus, recombinant protein expression.

• 12.
  Cell lysis:

  • a.
    Resuspend the cell pellet in 50 mL of lysis buffer (50 mM Tris pH 8, 250 mM NaCl, 5% glycerol, 3 mM 2-mercaptoethanol) (BioShop, Cat#SOD002, Cat#MER002) supplemented with lysozyme (100 μg mL⁻¹) (BioShop, Cat#LYS702). Rest on ice for 30 min.
  • b.
    Sonicate samples using a 5 s on and 15 s off pulse setting at 35% intensity for 6 min (Branson Sonifier, Model 450).
  • c.
    Pellet cellular debris via centrifugation at 39,000 g at 4°C for 30 min.
• 13.
  Prepare amylose resin (NEB, Cat#E8021L):

  • a.
    Pack amylose resin (0.6 mL bed volume) in a gravity flow column (Bio-Rad, Cat#7321010) and wash 5x column volume with ddH₂O.
  • b.
    Equilibrate resin by washing amylose resin 5x column volume with lysis buffer.
• 14.
  Purify the recombinant HRP using amylose affinity chromatography:

  • a.
    Apply the clarified supernatant to prepared amylose resin and rock for 30 min at 4°C in a conical tube.
  • b.
    Load sample onto column and elute non-specifically bound proteins by collecting initial flow through and 3x column volume washes with lysis buffer.
  • c.
    Elute protein with lysis buffer supplemented with 10 mM maltose (15 mL elution volume) (BioShop, Cat#MAL211).
• 15.
  Verify purity via SDS-PAGE (9% acrylamide/bis-acrylamide) and Coomassie staining (BioShop, Cat#CBB555).

  Note: Additional purification steps such as ion exchange and size exclusion chromatography are protein-dependent and subject to SDS-PAGE results. If necessary, purity should be enhanced prior to concentrating with the centrifugal filter.

  • a.
    Concentrate purified recombinant HRP protein with a centrifugal filter (Millipore Sigma, Cat#UFC9050) to desired protein concentration.

    Note: Molecular weight limit of the centrifugal filter will vary depending on the respective protein size.

    Optional: Exchange buffer via successive rounds of centrifugal filtration.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Mouse anti-MBP (1/1,000)	Cell Signaling Technology	Cat#2396S; RRID:AB_2140060
Goat anti-mouse DyLight 680 (1/10,000)	Invitrogen	Cat#35518; RRID:AB_614942
Bacterial and virus strains
E. coli DH5α	Agilent	Cat#200231
E. coli BL21(DE3) RIPL	Agilent	Cat#230280
Chemicals, peptides, and recombinant proteins
Dimethyl sulfoxide (DMSO)	BioShop	Cat#DMS555
Tris	BioShop	Cat#TRS001
Dithiothreitol (DTT)	BioShop	Cat#DTT001
Glycerol	BioShop	Cat#GLY004
Sodium dodecyl sulfate (SDS)	BioShop	Cat#SDS999
Bromophenol blue	BioShop	Cat#BRO222
Sodium chloride	BioShop	Cat#SOD002
2-Mercaptoethanol	BioShop	Cat#MER002
Maltose	BioShop	Cat#MAL211
Coomassie Brilliant Blue G-250	BioShop	Cat#CBB555
Polyphosphate 700	Kerafast	Cat#EUI003
Sodium phosphate	BioShop	Cat#SPM400
MOPS	BioShop	Cat#MOP001
EDTA	BioShop	Cat#EDT002
Sodium bisulfite	Caledon Laboratories	Cat#7320-1
Skim milk powder	BioShop	Cat#SKI400
Tween 20	BioShop	Cat#TWN510
Sodium azide	Sigma-Aldrich	Cat#S2002
Critical commercial assays
NuPAGE gels (4%–12% Bis-Tris)	Thermo Fisher Scientific	Cat#NP0336BOX
NuPAGE running buffer	Thermo Fisher Scientific	Cat#NP0001
Oligonucleotides
Primers: see Table S4 (Neville et al., 2023).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

Materials and equipment

Lysis Buffer

Reagent	Final concentration	Amount
Tris	50 mM	3 g
NaCl	250 mM	7.3 g
Glycerol	5%	25 mL
2-mercaptoethanol (14.2 M)	3 mM	105 μL
ddH2O		475 mL
Total	N/A	500 mL

Adjust to pH 8 with NaOH (vary pH depending on the specific protein). Store at 4°C for up to 4 weeks.

Homemade NuPAGE running buffer (TEMED-Free)

Reagent	Final concentration	Amount
MOPS	50 mM	5.3 g
Tris	50 mM	3 g
EDTA (500 mM pH 8)	1 mM	1 mL
SDS (20%)	0.1%	2.5 mL
Sodium bisulfite	5 mM	0.26 g
ddH2O
Total	N/A	500 mL

Store at 4°C for up to 4 weeks.

No salt buffer (NuPAGE mimetic)

Reagent	Final concentration	Amount
MOPS	50 mM	2.6 g
Tris	50 mM	1.51 g
EDTA (500 mM pH 8)	1 mM	0.5 mL
Sodium bisulfite	5 mM	0.52 g
ddH2O		249.5 mL
Total	N/A	250 mL

Adjust to pH 7.5 with Tris (vary pH and salt concentration if performing pH and salt disruption experiments). Store at 4°C for up to 4 weeks.

Step-by-step method details

PolyP treatment

Timing: 45 min

This section includes the steps of polyP binding to recombinant histidine repeat proteins and preparation for NuPAGE analysis. This protocol can be performed with purified proteins or crude lysate (see small scale lysate expression and troubleshooting problem 1). The outlined polyP treatment protocol describes using polyP₇₀₀ at a 5 mM final concentration. 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

• 1.
  PolyP preparation.

  • a.
    Prepare a 100 mM stock solution of polyP₇₀₀ (Kerafast, Cat#EUI003).

    • i.
      Dissolve 10.2 mg polyP₇₀₀ in 1 mL of MilliQ water for a 100 mM stock.

Note: All polyP concentrations stated herein are in terms of P_i monomers (sodium polyphosphate formula weight of 102 g mol⁻¹, regardless of chain length).

• 2.
  PolyP Treatment.

  • a.
    Add polyP₇₀₀ (5 mM final concentration) to recombinant HRP sample (4–10 μg of purified protein). Mix thoroughly.
  • b.
    Incubate sample with polyP for 30 min at room temperature (20°C–25°C).
  • c.
    Denature protein samples by adding 2X Laemmli Buffer to sample at a 1:1 ratio and boil at 95°C for 5 min.

CRITICAL: During sample preparation it is important to include negative controls for each HRP sample (no electrophoretic shift). The addition of monomeric NaH₂PO₄ (5 mM final concentration) (BioShop, Cat#SPM400) can be used to resolve the baseline apparent molecular weight of the protein sample in the absence of polyP₇₀₀. To confirm that polyP binding is histidine-dependent, additional controls must be included such as protein samples with deletions or mutations of the histidine repeat regions (see “the before you begin” section: deletion or mutagenesis of histidine repeat regions section).

Note: The buffer composition in which proteins are prepared for electrophoresis has negligible effect on NuPAGE shift. Even a sample in buffer of ionic strength/pH that would be prohibitive for HPM in solution (i.e., as observed via SEC or pull-down) will still yield a NuPAGE shift if the protein is indeed an HPM target. Thus, proteins in lysis buffer (50 mM Tris pH 8, 250 mM NaCl, 5% glycerol, 3 mM 2-mercaptoethanol) can be tested directly on NuPAGE without dialysis. This is likely because salt ions immediately separate from the relatively large protein and polyP molecules in the early stages of electrophoresis, allowing for HPM to occur in situ within the low-salt pH 7.5 conditions of the gel.

NuPAGE analysis

Timing: 2 days (2 h if using Coomassie stain)

This section includes the steps for HPM detection. PolyP-dependent electrophoretic mobility shifts are resolved on NuPAGE gels and visualized via western blot or Coomassie staining (Figure 1B).

• 3.Load samples onto precast 4%–12% Bis-Tris NuPAGE gel (Thermo Fisher Scientific, Cat#NP0336BOX) and run at 200 V for 45 min in 1X NuPAGE running buffer (Thermo Fisher Scientific, Cat#NP0001).

Alternative: Use a homemade equivalent (50 mM Tris, 50 mM MOPS, 1 mM EDTA, 5 mM sodium bisulfite and 0.1% SDS) (BioShop, Cat#MOP001 and Cat#EDT002, Caledon Laboratory Chemicals, Cat#7320–1).

Note: Traditional SDS-PAGE gels are unable to resolve polyP-dependent electrophoretic shifts likely due to tetramethylethylenediamine (TEMED), a polymerizing agent that is proposed to interfere with polyP chains bound to their targets. For more details see Bentley-DeSousa et al., (2018) and Baijal, K and Downey, M. (2021).^2,4

• 4.
  Detection of electrophoretic shift using western blot:

  • a.
    Transfer protein from NuPAGE gel to Immobilon-FL PVDF membrane (Millipore Sigma, Cat#IPFL00010) using a Trans-Blot Turbo Transfer System (Bio-Rad, Cat#1704150).

    • i.
      Activate PVDF membrane in methanol for 1 min prior to use.
    • ii.
      Set up transfer rig and run for 30 min at 5 V and 1.0 A.

      Optional: Observe total relative protein and success of transfer using Ponceau S Staining Solution (Thermo Fisher Scientific, Cat#A40000278).

  • b.
    Transfer the membrane to 1X Tris-buffered saline (TBS) (20 mM Tris, 150 mM NaCl, pH 7.4) for 5 min. Then proceed to incubate membrane with blocking solution (5% milk powder in TBS, 0.05% Tween-20 (TBST)) (BioShop, Cat#SKI400, and Cat#TWN510) for 1 h rocking at 4°C.
  • c.
    Antibody staining:

    • i.
      Pour off blocking solution and wash the membrane 2 × 5 min with TBST.
    • ii.
      Incubate membrane with primary antibody, mouse anti-MBP (1/1,000) (Cell Signaling, Cat#2396S) rocking for 18 h at 4°C.
    • iii.
      Pour off primary antibody and wash 3 × 5 min with TBST. Replace TBST with diluted secondary antibody, goat anti-mouse DyLight 680 (1/10,000) (Invitrogen, Cat#35518) and incubate for 1 h rocking at room temperature (20°C–25°C).

      Optional: Primary antibody (mouse anti-MBP) can be stored in blocking solution (supplemented with 0.02% sodium azide) (Sigma Aldrich, Cat#S2002) at 4°C and re-used for up to 8 weeks.

• 5.
  Analyze electrophoretic shifts:

  • a.
    Pour off secondary antibody and wash 3 × 5 min in TBST.
  • b.
    Image western blot on an Odyssey Li-Cor CL-x scanner (Li-Cor Biosciences).

    • i.
      Set LI-COR scan controls to have a resolution of 169 μm, medium scan quality and a focus offset of 0.0 mm.

Note: HPM targets are classified on the basis of a polyP-dependent increase in a protein’s apparent molecular weight (decreased mobility in the NuPAGE gel matrix).

Alternative: When testing purified proteins, electrophoretic shifts can be visualized with Coomassie stain (skip western blot steps, 4–5). Incubate NuPAGE gel with Coomassie stain for 15 min, then replace with a destaining solution (10% methanol, 10% acetic acid, 80% water) until protein bands become visible.

Size-exclusion chromatography (SEC)

Timing: 3 h per sample

This section includes steps to analyze polyP binding to purified HRP samples in solution. This technique allows for investigation of the effects of ionic strength and pH on HPM. SEC analysis was performed on MBP-MafB (80–232) and MBP-MafB ΔHis (80–131, 167–232) (Figure 2).

• 6.
  Equilibrate Superdex 200 Increase 10/300 GL size exclusion column (Cytiva, Cat#28990944):

  • a.
    Flow through two column volumes of a no-salt column buffer (50 mM MOPS, 50 mM Tris, 1 mM EDTA, 5 mM sodium bisulfite, pH 7.5) that mimics the NuPAGE gel running buffer.

CRITICAL: Filter and de-gas buffers using a 0.2 μm filter membrane to avoid air bubbles and particulate matter from obstructing column flow path.

• 7.
  Fractionate samples by FPLC using a Superdex 200 Increase 10/300 GL size exclusion column:

  • a.
    Inject a sample volume of 0.5 mL (10 μM protein) that has been pre-treated with polyP₇₀₀ (5 mM), see polyP treatment section.

    Note: Include negative controls (no polyP treatment, histidine deletion or mutation constructs, MBP alone) as separate runs.

    CRITICAL: Protein aggregation is detrimental to the size exclusion column. Perform a high-speed spin of protein samples at 9,400 g at 4°C for 10 min prior to loading samples onto the size exclusion column. Moreover, keep size exclusion instruments in a cold cabinet at 4°C to minimize further protein precipitation during the run.

  • b.
    Set a flow rate of 1 mL/min and collect 0.5 mL fractions for the total column volume.
  • c.
    Record A280 nm chromatograms to monitor elution profiles. Normalize absorbance values if there are variations in protein concentration.
• 8.
  Perform pH and salt disruption experiments:

  • a.
    Repeat fractionation of protein samples over a range of salt and pH conditions, modifying the composition of column buffer accordingly.

    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

  • b.
    Compare relative elution volumes using relative absorbance values and by running fractions on a NuPAGE gel.

Immobilized HRP polyP pull-down

Timing: 5 h

The MBP tag can also be leveraged to immobilize HRP candidates on amylose resin beads for polyP pull-down assays. This technique complements SEC for investigating the influence of ionic strength and pH on HPM.

• 9.
  Prepare HRP bound to amylose resin:

  • a.
    Directly continue from step 14a in the “large-scale expression and purification” section.

    Alternative: Begin from a liquid sample that was purified via amylose purification, ensure any maltose has been removed via exchange into fresh lysis buffer then rock with amylose resin for 30 min at 4°C.

    Note: Removal of maltose is required to rebind purified MBP-tagged protein onto amylose beads.

  • b.
    Wash the protein-bound resin with 3x volumes of the experimental test buffer (varied pH and/or ionic strength; based on the NuPAGE mimic recipe). To wash, pellet resin at 1000 g at 4°C for 1 min and pipette to discard supernatant.
  • c.
    Resuspend the washed resin in an equal volume of test buffer. Aliquot 19 μL of this slurry per test condition into 0.5 mL microtubes (4–10 μg of protein).
• 10.
  PolyP treatment.

  • a.
    Add the desired concentration of polyP or monomeric phosphate to each test tube.

    Note: We typically use 5 mM final polyP, in which case 1 μL of 100 mM polyP is added to the 19 μL slurry.

  • b.
    Incubate for 30 min at room temperature (20°C–25°C).
• 11.
  Washing.

  • a.
    Wash 4 × 500 μL with test buffer. For each wash, add buffer, gently vortex to suspend resin, then centrifuge at 1000 g at 4°C for 1 min. Discard supernatants.
• 12.
  Elution and analysis.

  • a.
    To each sample tube containing pelleted resin, add 20 μL of 2X Laemmli Buffer diluted 1:1 with MilliQ water. Vortex briefly. Boil at 95°C for 5 min to release proteins from beads.
  • b.
    Load 15 μL of eluate to NuPAGE gel and run as described in the NuPAGE analysis section.

CRITICAL: Only conditions that are permissive for polyP binding will result in a NuPAGE shift since unbound polyP is removed during the wash steps.

Note: This pull-down technique can also be used for removing unbound polyP prior to enzymatic assay of the modified protein. Simply swap the final Laemmli buffer elution step for an elution using test buffer supplement with 10 mM maltose.

Expected outcomes

This protocol can be used to identify targets of HPM. The extent of a polyP-dependent electrophoretic shift seen by NuPAGE analysis (the basis of identifying HPM candidates) is both chain length and concentration-dependent. HPM targets treated with polyP concentrations and chain lengths as low as 0.1 mM and 60 Pi units, respectively, tend to yield faint electrophoretic shifts that appear as smearing on the NuPAGE gel.¹ This smearing effect is likely due to incomplete polyP binding, whereby only a portion of the protein sample is polyP-bound. In contrast, as the chain length and concentration increase, the electrophoretic shift will sharpen and dramatically increase apparent molecular weight. These polyP-dependent electrophoretic shifts typically plateau at 5–20 mM polyP. While this modification has been shown to be histidine dependent, the minimum consensus sequence (number of histidine residues required to yield polyP-binding) remains elusive, thus, it is difficult to predict HPM candidates prior to NuPAGE analysis. Uniquely, HPM is a non-covalent ionic interaction that is strong enough to withstand denaturing conditions. Given its ionic nature, HPM is sensitive to pH and ionic strength, making it important to test variations of these parameters via the SEC and pull-down protocols as described herein.

Limitations

Histidine-tagged proteins cannot be used to analyze HPM. PolyP binding is histidine dependent, and a poly-His tag will introduce exogenous histidine residues to the protein sequence that can interact with polyP, see Figures S1A–S1C (Neville et al., 2023).¹ Alternative tags such as an MBP tag can be used, as well, endogenous histidine residues within the HRP can be leveraged and purified using immobilized metal affinity chromatography.

NuPAGE shifts typically require larger concentrations of polyP than those observed in most mammalian cells, which makes it difficult to ascertain physiological relevance of positive hits.

HRPs expressed recombinantly in E. coli may lack endogenous post translational modifications such as glycosylation, which could potentially influence HPM.

Troubleshooting

Problem 1

In the “before you begin” section, the crude lysates obtained from the small-scale expression will be difficult to pipette and load onto a NuPAGE gel. During the lysis protocol (Small-scale expression of HRPs: step 10b) an abundance of genomic DNA is released and likely causes the cell lysates to become extremely viscous.

Potential solution

Resuspend the cell pellet in 600 μL of lysis buffer (50 mM Tris, 250 mM NaCl, 5% glycerol, 3 mM 2-mercaptoethanol), then add Triton x-100 (0.1% final concentration) and 4 μL of lysozyme (50 mg/mL). Rest on ice for 30 min prior to sonicating the sample for 3 × 6 s at 35% intensity (Branson Sonifier, Model 450).

Problem 2

Protein aggregates and elutes in the void volume during SEC experiment. Unable to observe a shift in the elution volume upon polyP treatment (Size exclusion chromatography, steps 7–8).

Potential solution

The use of an MBP tag tends to largely enhance protein solubility; however, the TEV cleavage site introduces some instability to the protein when aberrant TEV site cleavage occurs. Therefore, removal of the MBP tag may help restore solubility in some cases. MBP-tagged HRP can be cleaved with TEV protease (0.02 mg/mL) for 18 h at 4°C while dialyzing against lysis buffer.⁵ The MBP tag can be removed by fractionation during the size exclusion experiment or by loading samples onto amylose resin prior to performing the SEC experiment. Alternatively, removal of the TEV-site during cloning may also help stability.

Other de-aggregation strategies include screening conditions that minimize aggregation such as screening buffers, protein concentrations and additives. As well, some HPM protein targets may require re-cloning and purifying smaller truncations that include the histidine repeat regions while omitting intrinsically disordered or unstable regions (Figure 2B).

Additionally, a different sized column may be required. We use the following columns, Superdex 200 Increase 10/300 GL and Superdex 200 16/600 (Cytiva, Cat#28990944 and Cat#28989335, respectively).

Problem 3

Multiple peaks are observed on size exclusion chromatography, which may indicate contamination, degradation or multiple oligomeric states (Size exclusion chromatography, steps 7–8).

Potential solution

Further purify the protein sample by conducting an additional SEC run prior to the analytical SEC run. Using the same conditions outlined above for SEC, load a concentrated protein sample, and collect fractions. Pool only the fractions judged as pure via SDS-PAGE stained with Coomassie blue. Use this purified protein for HPM testing. If there are multiple oligomeric states, fractions will need to be run on a NuPAGE gel to discern polyP shifts.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Zongchao Jia (jia@queensu.ca).

Technical contact

Questions about the technical specifics of performing the protocol should be directed to and will be answered by the technical contact, Dr. Zongchao Jia (jia@queensu.ca).

Materials availability

Plasmids generated in this study will be available upon request from the lead contact with a completed Materials Transfer Agreement.

Data and code availability

This study did not generate datasets or code.