Adapting recombinant bacterial alkaline phosphatase for nucleotide exchange of small GTPases

Abstract

The small GTPase Rat sarcoma virus proteins (RAS) are key regulators of cell growth and involved in 20–30% of cancers. RAS switches between its active state and inactive state via exchange of GTP (active) and GDP (inactive). Therefore, to study active protein, it needs to undergo nucleotide exchange to a non-hydrolysable GTP analog. Calf intestine alkaline phosphatase bound to agarose beads (CIP-agarose) is regularly used in a nucleotide exchange protocol to replace GDP with a non-hydrolysable analog. Due to pandemic supply problems and product shortages, we found the need for an alternative to this commercially available product. Here we describe how we generated a bacterial alkaline phosphatase (BAP) with an affinity tag bound to an agarose bead. This BAP completely exchanges the nucleotide in our samples, thereby demonstrating an alternative to the commercially available product using generally available laboratory equipment.

Introduction

The hydrolysis of nucleotide triphosphates underlies a variety of biological processes from DNA replication to cell growth [1, 2]. Often the nucleotide occupancy state of an enzyme is critical to its function [3, 4]. A well-studied example is that of the family of small GTPases and the notable sub-group of RAS GTPases. These enzymes are involved in the regulation of cell growth as they activate downstream pathways by interacting in their active GTP-loaded state with effector proteins, typically kinases [5, 6]. As such, mutations in RAS are implicated in 20–30% of all cancers and are a focus of much study for the development of therapeutics [7]. RAS proteins are inactive when GDP is bound in the nucleotide binding site and undergo a conformational change when acted upon by guanidine nucleotide exchange factors (e.g. SOS) to replace the GDP with GTP (Figure 1A) [8, 9]. The GTP-bound active state interacts with effectors and is a required reagent for many experiments in both structural biology and drug development [10–12]. Thus, the ability to produce these enzymes in a stable active state using a non-hydrolysable analog, is essential (Figure 1B).

Figure 1.

RAS Activation Cycle

A. Schematic of the RAS activation/inactivation cycle. B. Molecule representation of the nucleotides GTP and its non-hydrolysable homolog, GppNHp.

Recombinant RAS proteins typically have GDP bound when purified and protocols for exchanging these proteins to a GTP-bound active state center around the use of specific buffer conditions to reduce the affinity of the enzyme for the GDP and the addition of a phosphatase to further convert the released GDP to GMP so that it cannot reenter the active site [13]. However, only a few commercial phosphatase products are available for this assay, and this has consequences as was seen during supply chain disruptions during the COVID-19 pandemic. Recent advances in the production of recombinant BAP allow for cytosolic production instead of targeting the protein to the periplasm [14–16], which increases the yield and simplifies production of this reagent [17].

We exploited these improvements to produce a modified BAP with an affinity tag for both initial purification and as a means of removing BAP protein from the completed reaction. Further, by pre-binding the protein to the resin, the protein can be used in the same manner as the commercial Calf Intestinal Phosphatase product.

Methods and Materials

Cloning and Expression

Cloning of GG-Hs.KRAS(2–185), GG-Hs.KRAS(1–169), GG-Hs.KRAS(2–169) was previously described [18, 19]. DNA encoding for amino acids (22–471) of the PhoA gene was synthesized by ATUM (Newark, NJ) as a Gateway Entry clone optimized for E. coli expression. The Entry clone was sub-cloned into pDest-521 (Addgene # 159688), a Gateway vector containing an aminoterminal His6 tag to produce His6-Ec.BAP (22–471) (Figure 2A). The BL21 STAR E. coli strain containing the DE3 lysogen and rare tRNAs (pRare plasmid, Cm^R) (Invitrogen cat # C601003) was transformed with the target expressing plasmid (Amp^R) and used for protein production. Expression was done as previously described [20]. Briefly, an overnight E. coli seed culture was started by inoculating 50 mL of MDAG medium [20] in a 250 mL baffled shake flask with transformed cells from a glycerol stock and incubating the flask for 16 hours at 37°C until mid-log phase growth. Ten mL (2% of production volume) of the seed culture was used to inoculate 500 mL of Dynamite broth [20] in a 2 L baffled shake flask. The culture was grown at 37°C and 250 RPM using a one-inch orbit shaker (Eppendorf). When the OD₆₀₀ reached 6.0–8.0 (~5 hours) the culture was chilled to 16°C and IPTG was added to a final concentration of 0.5 mM. The culture was grown overnight (~17 additional hours), and the cells collected by centrifugation at 3990 × g for 20 minutes. Cell pellets were immediately frozen at −80°C [20].

Figure 2.

A. A representation of the His6-BAP protein purified in this work. B. Representative SEC chromatograms of the A280 nm trace from a 16/600 Superdex 200 for His6-BAP protein. Blue trace is the target protein and brown traces are molecular weight standards (Bio-Rad cat # 1511901). C. A line graph representation of the BAP activity assay data (nmoles/min vs PhoA concentration. D. Coomassie-stained SDS-PAGE of the purified BAP protein. 1-Invitrogen Benchmark protein ladder, 2-one μg BAP, 3-five μg BAP.

Purification

GG-Hs.KRAS(2–185), GG-Hs.KRAS(1–169), GG-Hs.KRAS(2–169) were purified as previously described [21]. Purification of His6-BAP was performed as described in Krawczun et al. with several alterations. The lysis buffer was altered to 20 mM Tris pH 8, 300 mM NaCl, 5 mM TCEP, 10% glycerol (v/v), and 0.02% triton X-100. Following immobilized metal affinity chromatography (IMAC), the protein was purified over a HiLoad 26/600 Superdex 75 pg (Cytiva) using buffer containing 20 mM HEPES, pH 7.4, 100 mM KCl, 50 mM NaCl, 0.2 mM MgCl₂, 0.2 mM ZnCl₂, 10% glycerol (v/v), 0.02% triton X-100, and 0.05% Tween20. The final sample of His-BAP was applied to an analytical SEC column (Superdex 200 Increase 10/300 GL, Cytiva, cat # 28990944) using the same buffer. [17].

Analysis of BAP activity with p-Nitrophenyl phosphate (pNPP)

BAP activity was assessed by calculating the rate of hydrolysis of pNPP with a Unit defined as the amount of enzyme that will hydrolyze 1 nmole of pNPP in 1 minute at room temperature. The assay was performed in a total volume of 50 mL in 20 mM HEPES, pH 7.3, 150 mM NaCl, 1 mM TCEP, and 50 mM pNPP. The assay was performed on a serial dilution of the BAP at 40 mM, 20 mM, 10 mM, 5 mM, 2.5 mM, 1 mM, and 0.5 mM for 10 minutes. The reaction was stopped by the addition of 1 mL of 1 N NaOH. Results were read on a FLUOstar Omega plate reader at 405 nm. The assay was performed twice.

Nucleotide Exchange with BAP

BAP was substituted for commercial CIP-agarose (Millipore Sigma cat# P0762) in our exchange assay [22]. Three reactions were set up by combining 2 mg, 1 mg, and 0.5 mg of BAP with 1 mg of GG-Hs.KRAS(2–185) at a final concentration of 95 mM and GppNHp (Guanosine-5'-[(β,γ)-imido]triphosphate, tetralithium salt, Jena Biosciences, cat # NU-401–50) at a 10x molar ratio to the protein in 20 mM HEPES, pH 7.3, 150 mM NaCl, 1 mM TCEP, 0.1 mM ZnCl₂, and 200 mM ammonium sulfate. The final reaction volume was 500 μL. Reactions were mixed at room temperature for 3 hrs, and then each reaction was loaded on a 1 mL IMAC column (Cytiva, HiTrap IMAC HP, cat # 17524701) at 1 mL/min in 20 mM HEPES, pH 7.3, 150 mM NaCl, 1 mM TCEP. The columns were washed with 4 mL of the same buffer, and then with the same buffer containing 30 mM and 500 mM imidazole. Five hundred μL fractions were collected throughout the run. Five μL of 50 mM GppNHp and 2.5 μL of 1 M MgCl₂ was added to each fraction at the end of the run. Peak fractions (based on stained gel or A₂₈₀ signal) were pooled and applied to PD-10 columns (Cytiva, cat # 17085101) equilibrated in 20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM MgCl₂, and 1 mM TCEP, to remove excess nucleotide. Due to some carryover contamination of the protein with nucleotide, the standard PD-10 protocol from the manufacturer was modified by limiting the load volume to 1.5 mL. The elution was collected in 0.5 mL fractions to be analyzed by A₂₈₀ readings. All resultant pools were tested for nucleotide loading.

HPLC analysis of bound nucleotide

Ion-Pairing Reversed Phase HPLC analysis of protein-bound nucleotides was done by preparing individual 1 M stock solutions of K₂HPO₄ and KH₂PO₄. Two liters of 100 mM K₂HPO₄/KH₂PO₄, pH 7.2 solution was prepared by adding 143.4 mL of 1 M K₂HPO₄ to 56.6 mL of 1 M KH₂PO₄ and bringing to a volume of 2 L with dH₂O in a 2 L graduated cylinder. A 4 mM tetrabutyl ammonium hydrogen sulfate (TBAHS) solution (Buffer A) was prepared by mixing 250 mL of TBAHS with 750 mL of the 100 mM K₂HPO₄/KH₂PO₄ buffer_. Buffer B was prepared by mixing 210 mL of Buffer A with 90 mL of acetonitrile. A Waters e2695 Alliance System with Empower3 software was primed with Buffers A and B, and equilibration of a Hichrom Ultrasphere 5 μm C18 ODS HPLC Column, 4.6×250 mm was done at a 50% A/B buffer mixture for 1 hour, followed by a 98% A buffer mixture for 2 hours. Nucleotide standard mixtures were prepared at 1 and 10 mM with the appropriate nucleotides. Samples were diluted 1:10 in Buffer A prior to analysis. All samples were analyzed at a flow rate of 0.6 mL/min, using 98/2% A/B buffer for 5 minutes, followed by a 27 minute gradient to 65/35% A/B buffer, held for 2 minutes, and then returning to 98/2% A/B buffer to re-equilibrate the column.

BAP preparation on IMAC beads

One mL of a 50% slurry of Ni Sepharose High Performance resin (Cytiva, cat # 17526802) was prepared for protein binding by washing the resin with water, followed by several washes with 20 mM HEPES, pH 7.3, 150 mM NaCl, 1 mM TCEP. To this resin 10 mg of His6-BAP was added and batch mixed for 2 hours at room temperature. The beads were separated from the non-binding material by briefly centrifuging the samples at 1000 × g. The resin was washed three times with 20 mM HEPES, pH 7.4, 150 mM NaCl, 1mM ZnCl₂, 200 mM ammonium sulfate, and stored as an approximate 50% slurry, at 4°C.

Nucleotide exchange with BAP on IMAC beads

To test the activity of the BAP bound to the IMAC beads, four 1 mL reactions were set up with a dilution series of the 50% slurry at 4 μL, 8 μL, 16 μL, and 32 μL. The reactions contained 2 mg of GG-Hs.KRAS4b(2–169) each and GppNHp at a 10x molar ratio to the protein in 20 mM HEPES, pH 7.3, 150 mM NaCl, 1 mM TCEP, 0.1 mM ZnCl₂, and 200 mM ammonium sulfate. Reactions were mixed for 3.5 hours at room temperature. The resin was briefly centrifuged at 1000 × g and the sample (supernatant) removed with a pipet. A 20x molar excess of GppNHp was added to the sample and MgCl₂ was added to 5 mM and the sample was incubated at 4°C overnight. Samples were desalted over PD-10 columns and analyzed by HPLC as outlined above for bound nucleotide. The BAP/IMAC slurry was stored at 4°C. After four months, the exchange procedure was repeated to check the stability of the enzyme, using 8 μL of slurry to 2 mg of KRAS. Throughout all procedures, protein concentrations were measured spectrophotometrically with a Thermo Scientific NanodropOne.

Intact mass

Protein samples were diluted to 0.1 mg/ml in a total volume of 50 μL using 5% acetonitrile/0.2% formic acid (Buffer A). The samples were transferred to a macrovial (Thermo Fisher Scientific cat # C4011-LV12) and 2 μL was analyzed via liquid chromatography coupled on-line with mass spectrometry. Reversed-phase separation was performed on a Vanquish UHPLC chromatographic system using a MabPac HPLC Column (Thermo Fisher Scientific, cat # 088645) maintained at 50°C. Samples were loaded onto the column at 500 μL/min at 2% Buffer B (47.5% acetonitrile/47.5% isopropanol/0.2% formic acid) for 30 sec. Protein was eluted from the column in a gradient from 2% B to 100% B over 5 minutes followed by a wash for 2.5 minutes at 100% Buffer B. The column was then equilibrated at 2% Buffer B for 2 minutes. Proteins were ionized with an API Source coupled to an Exactive Plus extended mass range orbitrap mass spectrometer (Thermo Fisher Scientific). High-resolution intact protein mass (MS1) spectra were acquired over a 600–2500 m/z window at 120,000 FT resolution (at 400 m/z) with an AGC target value of 3e+06 and averaging 4 microscans. Spectra were then analyzed by MagTran (Amgen Inc.)

Results and Discussion:

Purification and Activity

Modifying the nucleotide state of proteins is essential for laboratories like ours, and the reagents involved in this can become limiting and financially burdensome. Therefor it is incumbent for us to seek out in-house production. Recent improvements surrounding BAP purification [17] led us to replicate the CIP-agarose reagent that we regularly use. Purification of His6-BAP by IMAC, even with our buffer adjustments, proceeded in a similar manner to Krawczun et al. SEC analysis showed the purified protein to be in a tetrameric state (Figure 2B), SDS-PAGE indicated purity of >95% (Figure 2C), and the final yield was 190 mg/L. Contrary to the published results in Krawczum et al., we did not need to go through an activation step as our initial testing proved that the protein was active directly out of the purification process. The activity analysis by pNPP digestion generated a rate of digestion calculation of 8.74 U (nmols/min/ug) (Figure 2D). These results indicated to us that we could make active BAP in a cost-effective manner.

GppNHp exchange

To determine if the purified His6-BAP was functioning in our nucleotide exchange procedure, we performed nucleotide exchange of a common protein in our lab, GG-Hs.KRAS(2–185), which encompasses the G-domain and hypervariable region of this protein. In all three tests, the proteins were completely exchanged, and no GDP was detected (Figure 3A). However, in order to seamlessly substitute the His6-BAP for the commercial CIP-agarose beads, and to avoid unnecessary purification over an IMAC column to get rid of His6-BAP, His6-BAP was bound to IMAC beads. His6-BAP bound efficiently to IMAC resin with only 5% of the protein failing to bind the resin (data not shown), giving us confidence that beads were saturated.

Figure 3.

A. A representative HPLC analysis of the nucleotide stripped from the KRAS4b after exchange with GppNHp utilizing the His6-BAP reagent in the reaction. The retention times of the nucleotide standards (indicated by arrows) were consistent both before and after application of the test sample. B. Intact mass analysis. Deconvoluted data analysis (Magtran) of the exchanged KRAS4b. Predicted MW of GG-Hs.KRAS4b(2–169) and His6-BAP are 19.2 kDa and 48.3 kDa, respectively.

Validation of bead-bound His6-BAP

We next tested the suitability of replacing the CIP-agarose with the IMAC bead-bound His6-BAP to exchange the bound GDP for GppNHp at four different concentrations. In this experiment, we used a common lab reagent, GG-Hs.KRAS4b(2–169), commonly referred to as the KRAS4b G-domain. Again, we used the same HPLC analysis technique of the nucleotides bound to the KRAS4b after the nucleotide exchange, which indicated that GppNHp was the only nucleotide bound to the protein (Figure 3A), even at the minimal amount of 4 μL of slurry to 2 mg of KRAS4b. To test another aspect of our standard use of CIP-agarose, we repeated the exchange reaction with His6-BAP-IMAC beads that were stored at 4°C for four months. Again, the only nucleotide detected in the exchanged sample was GppNHp (Figure 3A), demonstrating the stability of the enzyme. There was some concern that His6-BAP might leach from the IMAC beads during the protocol. However, no signal was observed at the predicted molecular weight of His6-BAP (48,322 Da) by electrospray ionization mass spectrometry of the final sample (Figure 3B). This suggests that no His6-BAP leached from the beads.

Conclusions

Often a single reagent can become rate limiting to a project. In our case the phosphatase needed to perform nucleotide exchange is such a reagent. In this work we have outlined the protocols need to produce His6-BAP bound to IMAC beads and is easily created in the laboratory at high yield and can be incorporated seamlessly into existing nucleotide exchange protocols with no reduction in exchange efficiency. Laboratories that routinely perform nucleotide exchanges of small GTPases such as the RAS proteins, will likely have the capability to easily produce this reagent at significantly reduced cost compared to commercial alternatives.

Highlights.

• Detailed in-house production protocol for making resin bound bacterial alkaline phosphatase (BAP)

• Cheap and reliable reagent to replace commercial agarose bead conjugated phosphatases

• Nucleotide exchange protocols for small GTPase’s such as Rat sarcoma virus proteins (RAS)