Expression and purification of Steroidogenic Factor-1 (NR5A1) nuclear receptor ligand binding domain complexed with the signaling phosphoinositide lipid PI(3,4,5)P3

Abstract

Steroidogenic Factor-1 (SF-1, NR5A1) is an orphan nuclear receptor that plays a critical role in proper development and adult function of the adrenals, gonads and the ventral medial hypothalamus in the brain. Nuclear Structural studies have revealed that the SF-1 ligand-binding domain is capable of interaction with glycero-phospholipids, such as phosphoinositides, in its binding pocket. Of these, the phosphoinositide PI(3,4,5)P3 (PIP3) has a high affinity for SF-1 and functions as a key regulatory ligand. This paper details the methods for the recombinant expression and purification of the SF-1 protein, as well as a novel protocol for isolating pure SF-1 bound to PIP3. These methods are a foundational step for future biophysical assays, crystallography studies, and the development of targeted therapeutics for SF-1-dependent adrenocortical carcinoma.

2. Introduction

Steroidogenic Factor-1 (SF-1, NR5A1) is an orphan nuclear receptor that functions as a transcription factor, playing a key role in the proper regulation of gene expression, steroidogenesis, sex determination and adrenal gland development and adult functions¹. Critical functions of SF-1 in development have been highlighted in mice studies showing developmental knockout of SF-1 causes death around postnatal day 8 due to a complete lack of corticosteroid production, as both males and females do not have adrenal glands^2,3. These mice can be rescued by adrenal transplants and perinatal corticosteroid injections, but typically develop an obese phenotype⁴ . Conversely, overexpression of SF-1 has been linked to adrenocortical carcinoma, and the proliferation of these cells decreases when inhibiting SF-1⁵.

The ligand binding domain (LBD) of SF-1 is crucial for its regulation, as this is classically where nuclear receptors interact with regulatory ligands³. Published crystal structures of the LBD reveal a largely hydrophobic core of the pocket with a hydrophilic area near its opening^6–13.This structure helps regulate what endogenous ligands are able to bind to SF-1; the most well-characterized being the signaling phosphoinositide lipids PI(4,5)P2 and PI(3,4,5)P3 (PIP3)^14–21. It is known that the positioning of the acyl chains and headgroups of phosphoinositides repeats the same pattern in which the acyl chains follow the same path to reach deep within the hydrophobic core of SF-1, while the headgroup is exposed outside the pocket, in a highly solvent-exposed position^14,22.

The phosphoinositide PI(3,4,5)P3 (PIP3) has been found to bind with high affinity to SF-1, and has been suggested as a regulatory ligand of SF-1, verified with several crystal structures. These structures and follow-up functional work all suggest both the headgroups of bound phospholipids^{7,16,17,21,23,24} as well as the composition of the acyl chains¹⁸ can regulate SF-1, partly through distinct mechanisms^14,22. Levels of PIP3 in mammalian cells associate with activation of SF-1 function in gene regulation^15,25, dependent on the ability of SF-1 to bind phospholipids¹⁵. Although many structures now exist of SF-1 bound to various phosphoinositides, the technical path to expression and purification of these complexes has been anything but straightforward. Here, we present a standardized approach to expression and purification of SF-1, and how to complex SF-1 with the phosphoinositides PIP2 and PIP3, in order to use this as a reagent to further characterize the biophysical functions of these intriguing protein/lipid complexes.

3. Plasmid Construction, Transformation into Escherichia Coli, and Glycerol Stock Preparation

The design of a high-yield human SF-1 encoding cDNA is dependent on numerous factors, including but not limited to expression system, codon optimization, and the optimization of the final product. Many constructs have been used to express human and mouse SF-1 LBD, all expression and purification conditions are identical for human vs. mouse SF-1 LBD^7,8. A Cys-lite version (C247S, C412S) of human SF-1 LBD has been used successfully for crystallography spanning amino acids 218–461 of the human SF-1 sequence (RefSeq Transcript ID NM_004959.5, RefSeq Protein ID NP_004950.2) in a pET-based expression vector for production of His₆-containing proteins in standard BL21 DE3 bacterial expression systems (Figure 1).

Figure 1.

Plasmid construction of human SF-1 LBD. SF-1 LBD (218-461) plasmid design uses a pET-based backbone with a standard T7 promoter for IPTG induction, with an N-termal 6X-His tag and a Tev cleavage site. Table below explains the function of each element contained in the vector, image from Vector Bee, table adapted from information provided by VectorBuilder.

1. BL21-GOLD (DE3) cells and SF-1 LBD encoding plasmids were set on ice to thaw gradually.

2. Add 1 μl (~10 ng) plasmid DNA to the cell mixture. Carefully flick the tube 4–5 times to mix cells and DNA.

3. Place the mixture on ice for 30 minutes and allow the mixture to equilibrate

4. Heat shock at exactly 42°C for exactly 10 seconds on a heating block.

5. Place on ice for 5 minutes.

6. Pipette 950 μl of room temperature SOC into the mixture over a flame to avoid contamination.

7. Attach the tube to a shaker, set to 37°C, 200 rpm, for 60 minutes.

8. Warm selection plates (LA/Ampicillin) to 37°C.

9. Mix the cells thoroughly by repeatedly drawing in and expelling with a P200 micropipette.

10. Spread 100 μl onto a selection plate, allowing all liquid to dry before flipping the plate over, and incubate overnight at 37°C.

11. Select a single colony, and add to a 250 mL Furnbach flask containing 100 mL TB/ampicillin (100 μg/mL) overnight culture, shaking at 200 rpm, 37 °C.

12. Mix 750 μL overnight culture with 750 μL 50% glycerol (v/v). This stock should be stored at −80 °C.

4. Escherichia Coli encoded His₆-SF-1-LBD Expression and Purification

4.1. Equipment

• Refrigerated shaker-incubator

• Low-speed centrifuge, rotors, and 1 L bottles

• Ultrasonic Cell Disruptor

• High-speed centrifuge and rotor for 30-50 mL bottles

• ÄKTA pure protein purification system (Cytiva)

• Nanodrop spectrophotometer (ThermoFisher)

4.2. Materials

• His₆-SF-1 LBD encoded BL21-GOLD (DE3) bacterial cells

• 250 mL Fernbach flask with 100 mL Terrific Broth (TB) and 100 μg/mL ampicillin

• 4 L Fernbach flasks with 1 L Terrific Broth (TB) and 100 μg/mL ampicillin

• 1 M solution Sterile-filtered Isopropyl-β-D-thiogalactopyranoside (IPTG) in water

• Complete protease inhibitor tablets, EDTA-free (Roche)

• 10,000 MWCO Slide-A-Lyzer G3 Dialysis Cassette (ThermoFisher)

• Amicon Ultra Centrifugal Filters, 15 mL, 10,000 NMWL (MilliporeSigma)

• His₆-TEV

• Lysis Buffer: 50 mM HEPES pH 7.5, 300 mM KCl, 20 mM imidazole, 2 mM chapes, 5% (v/v) glycerol, 5 mM β-mercaptoethanol (β-ME)

• Elution Buffer: 50 mM HEPES pH 7.5, 150 mM KCl, 300 mM imidazole, 2 mM chapes, 5% (v/v) glycerol, 5 mM β-mercaptoethanol (β-ME)

• Buffer A: 50 mM HEPES pH 7.5, 2 mM chapes, 5 mM β-mercaptoethanol (β-ME)

• Buffer B: 50 mM HEPES pH 7.5, 1 M NaCl, 2 mM chapes, 5 mM β-mercaptoethanol (β-ME)

• Gravity columns (100 mL and 20 mL volumes)

• HisPur cobalt resin (ThermoFisher)

• Two HiTrap Desaltin 5 mL columns with Sephadex G-25 resin (Cytiva)

• HiTrap Capto Q 1 mL anion exchange chromatography column (Cytiva)

4.3. Growth Procedure

1. Using a sterile micropipette tip, a 250 mL Furnbach flask containing 100 mL TB/ampicillin (100 μg/mL) was inoculated with His₆-SF-1 LBD containing BL21-GOLD (DE3) cells from a previously made glycerol stock, and grown overnight at 37 °C.

2. A 10 mL aliquot of the overnight culture was added to each 4 L Furnbach flash containing 1 L TB/ampicillin (100 μg/mL). This is shaken at 37 °C, 200 rpm, until the OD₆₀₀ reaches 0.6, at which point the cultures are cooled by refrigeration for 10-20 minutes, and 1 mL of 1 M IPTG is added. The culture is then allowed to grow overnight at 18 °C, 180 rpm.

3. The cells are then centrifuged at 5000 rpm at 4 °C for 30-40 minutes, collecting the pellet.

4.4. SF-1 LBD Purification Protocol

1. All steps are completed at 4 °C.

2. Each liter of cell pellet is homogenized in 40-50 mL of lysis buffer and one cOmplete tablet, EDTA-free.

3. The rehomogenized cells are then sonicated in an ultrasonic cell disruptor for 10 minutes at 70 % amplification, 30% duty cycle (3 seconds on, 7 seconds off) on ice.

4. Lysed cells are then centrifuged at 15,000 rpm, 4 °C, for 30-40 minutes with the supernatant being collected.

5. The lysate is then nunated overnight with 0.5 mL HisPur Cobalt resin.

6. The lysate/HisPure cobalt resin is then added to a 100 mL gravity column, and the flowthrough is collected and removed.

7. The HisPure cobalt resin is then washed three times with 20-40 mL lysis buffer.

8. 25 mL of elution buffer is then added to the column and allowed to incubate for 30 minutes, at which point a slow elution is completed, collecting five 5 mL fractions.

9. His₆-TEV is added to the eluted SF-1 LBD at a ratio of 2 mg TEV: 50 mg His₆-SF-1 LBD) and loaded into a rehydrated 10,000 MWCO Slide-A-Lyzer dialysis cassette and dialyzed overnight in 2 L lysis buffer.

10. An SDS-PAGE is run to ensure complete cleavage of the poly-His tag (cleaved SF-1 LBD should run slightly lower on the gel).

11. The cleaved SF-1 LBD and TEV are then nunated for 30 minutes with 0.1 mL HisPure cobalt resin.

12. The sample is then loaded in a 20 mL gravity column and the flowthrough is collected.

13. The flowthrough is then desalted by two HiTrap Sephadex G-25 columns pre-equilibrated with buffer A, on an ÄKTA pure protein purification system by an isocratic flow of buffer A.

14. The eluted protein is then concentrated by centrifugation in a 10,000 NMWL filter unit at 3,000 rpm, 4 °C, to 4-5 mL.

15. The concentrated sample is then loaded onto a buffer A pre-equilibrated 1 mL HiTrap CaptoQ column on an ÄKTA pure protein purification system.

16. The loaded column is then equilibrated with 30-50 mL buffer A. SF-1 LBD is then eluted by a 0-50% gradient of buffer B (Figure 2).

17. If desired, the sample can be concentrated as described in step 14. At this point the sample can also be dialyzed into a buffer without Chapes.

18. The concentration of SF-1 LBD is determined by molar absorptivity at 280 nm by nanodrop (ε = 20,315 M⁻¹ cm⁻¹, 1.422 g/L)

Figure 2.

Chromatography Purification of SF-1 LBD. Anion Exchange Chromatography was used for initial purification of SF1-LBD by CaptoQ Cytivia Column. Featured in this figure is a chromatogram of percentage of Buffer B needed to elute off SF-1 LBD. Protein elution is identified by absorbance values (mAU).

4.5. Expected Outcomes

A typical yield of SF-1 LBD is at least 12.5 mg/L of culture, of properly folded SF-1 LBD is observed.

4.6. Advantages

This purification utilizes readily available and procedurally defined poly-his tag affinity chromatographic methods, which makes the procedure easily accessible and easily completed by trainees.

4.7. Cautions and Troubleshooting

• The TB must be cooled to < 25 °C before inoculation with IPTG

• Chapes must be maintained at a minimum concentration of 2 mM to prior to ion exchange chromatography.

5. Bacterial Phospholipid Exchange with PIP3 of SF-1 LBD

5.4. Equipment

• ÄKTA pure protein purification (Cytiva)

• Nanodrop spectrophotometer (ThermoFisher)

• Ultrasonic Bath 1.9L (Fisher Scientific)

5.5. Materials

• Phasphitdylinositol-3,4,5-triphosphate (Cayman Chemical)

• HiTrap Capto Q 1 mL anion exchange chromatography column (Cytiva)

• Two HiTrap Desaltin 5 mL columns with Sephadex G-25 resin (Cytiva)

• Buffer A: 50 mM HEPES pH 7.5

• Buffer B: 50 mM HEPES pH 7.5, 1 M NaCl

• Thin borosilicate glass tube (Fisherband)

5.6. PIP3 micelle Preparation

1. Reconstitute the vacuum-sealed PIP3 from Cayman Chemical to 1 g/L in Buffer A. Use a 1 mL syringe with at least a 22-gauge needle to pierce the septum and draw water into the glass vial. If lyophilized phosphoinositide is used as the starting material under vacuum, micelles result from this procedure. If a film of lipids at the bottom of the vial is used, multi-lamellar vesicles result, in which the vast majority of the lipids are inaccessible to SF-1 for binding, due to the multi-lamellar nature of the vesicles. This is why micelles are used, such that 100% of the lipid is available for binding to SF-1.

2. Sonicate the vesicle in a room temperature or heated water bath glass ware sonicator for at least 5 minutes to generate micelles. This sonication procedure should be repeated after micelles have rested for an hour, so resonicate the micelles often.

5.7. SF-1/bpL exchange with PIP3

1. Place the desired amount of SF-1/bPL previously desalted into Buffer, into a tall, thin borosilicate glass tube.

2. Add 1ug/uL of the prepared PIP3 solution dropwise to the surface of the SF-1 solution.

3. Mix the SF-1/PIP3 solution thoroughly using a P1000 pipette.

4. Carefully layer nitrogen gas over the reaction mixture before sealing the glass tube to minimize air-water interface.

5. Incubate the mixture at room temperature for 16 hours while gently nutating on a belly dancer or similar shaker.

5.8. Isolating SF-1/PIP3 by AIEX Column

1. Following the overnight incubation, load the sample onto a MonoQ 5/50 column.

2. Elute the protein using a gradient of Buffer B against Buffer A.

3. The SF-1/PIP3 complex is expected to elute later off the column than the unexchanged SF-1 bound to bPL (Figure 3). Pool the fractions corresponding to the SF-1/PIP3 fraction peak.

4. Add 2uL of freshly sonicated 1g/L PIP3 micelles to isolated SF-1/PIP3 to maintain complex stability.

Figure 3.

Chromatography Isolation of SF-1: PIP3. Anion Exchange Chromatography was used for collection of PIP3 bound SF1-LBD by MonoQ 5/50 Cytivia Column. Featured in this figure is a chromatogram of percentage of Buffer B needed to elute off SF-1:PIP3. Protein elution is identified by absorbance values (mAU)

5.9. Expected Outcomes

When a 3-molar excess of PIP3 to SF1 is incubated, a yield of 40-50% of SF-1 bound to PIP3 can be expected.

5.10. Advantages

This purification method allows for the isolation of pure SF-1 bound to PIP3, which can readily be used for downstream applications such as biophysical assays and crystallography.

5.11. Cautions and Troubleshooting

• It is ideal to minimize the air-water interface during the exchange, as PIP3 can potentially arrange itself at this interface inhibiting the exchange

• The exact molar ratio of PIP3 to SF-1 may need to be optimized to your specific protein batch as well as the acyl chain composition of PIP3 ligand used to achieve the highest yield.

• Before running columns ensure that the SF-1 protein is completely desalted into Buffee A before loading on the column to ensure the protein is able to stay on the columns.

6. Conclusions

While Steroidogenic Factor 1 (SF-1) is classified as an orphan nuclear receptor, in vitro analysis suggests that phosphoinositides such as PI(3,4,5)P3 (PIP3 ),may serve as key regulatory ligand. Understanding the mechanisms by which SF-1 is regulating biologically is imperative for the treatment of conditions like adrenocortical carcinoma. The protocol outline in this paper provides a simple ,reliable way to generate pure SF-1/PIP3 complexes for further study. These purified can be used to conduct both biophysical and structural assays. The information acquired from this further analysis can be leveraged to clarify the regulatory mechanism of SF-1, ultimately providing a targetable mechanism for the treatment of SF-1 dependent cancers such as adrenocortical carcinoma.