Protein expression, purification, crystallization and crystallographic studies of BPSL0741 from Burkholderia pseudomallei

The B. pseudomallei protein BPSL0741, which has previously been identified to be essential for survival of the bacterium in an infected host, was successfully overexpressed in its recombinant form and purified to homogeneity. The recombinant protein appeared to be monomeric in solution and the protein crystal diffracted to 2.1 Å resolution.

Abstract

Burkholderia pseudomallei is the causative agent of the lethal disease melioidosis. This bacterium infects animals and humans and is increasingly resistant to multiple antibiotics. Recently, genes associated with survival of the bacterium in the infected host have been identified. One of these genes, bpsl0741, is annotated as a hypothetical protein of 185 amino acids. Here, recombinant BPSL0741 (rBPSL0741) protein was expressed, purified, verified by mass spectrometry, crystallized and analyzed by X-ray diffraction. rBPSL0741 was crystallized by vapor diffusion using a reservoir solution consisting of 0.2 M ammonium acetate, 0.1 M sodium acetate trihydrate pH 4.6, 30% PEG 4000. The crystals diffracted to 2.1 Å resolution using an in-house X-ray diffractometer and belonged to an orthorhombic space group, with unit-cell parameters a = 62.92, b = 64.57, c = 89.16 Å. The Matthews coefficient (V_M) was calculated to be 2.18 ų Da⁻¹, suggesting the presence of two molecules per asymmetric unit and an estimated solvent content of 43.5%. The crystal was deemed to be suitable for further structural studies, which are currently ongoing.

1. Introduction

Burkholderia pseudomallei, a bacterial pathogen of humans and animals, causes the lethal disease melioidosis, which can result in high rates of morbidity and mortality (Nathan et al., 2018 ▸). Melioidosis is mainly endemic to Southeast Asia and Northern Australia, and is expanding into tropical and sub­tropical regions, including America, Africa and the Pacific and Indian Ocean island nations (Galyov et al., 2010 ▸; Schweizer, 2012 ▸; Meumann et al., 2024 ▸). Melioidosis patients are currently treated with a combination of antibiotics, but B. pseudomallei is gaining resistance to ampicillin and penicillin, as well as first-generation and second-generation cephalosporins. Furthermore, no vaccine is available to treat this disease, leading to limited treatment options (Wiersinga et al., 2018 ▸). B. pseudomallei has been categorized as a Tier 1 select agent by the Centers for Disease Control and Prevention (CDC), USA (Peacock et al., 2008 ▸).

The first reference genome of B. pseudomallei was for strain K96243 from Thailand and comprised two replicons of 4.07 Mb (chromosome 1) and 3.17 Mb (chromosome 2) (Holden et al., 2004 ▸). Chromosome 1 carries genes associated with cell growth and metabolism, while chromosome 2 contains genes essential for the flexibility of this bacterium to survive in different environments (Wiersinga et al., 2006 ▸). At the time that the genome sequence was released, 25% of its coding DNA sequences (CDSs) were annotated as hypothetical proteins (HPs; Holden et al., 2004 ▸). Previous studies have suggested that HPs may play important roles as they make up a significant portion of proteomes. The significant number of HPs encoded by genomes strongly suggests the novelty of these proteins, where the unknown biological functions of these HPs may play key roles in the physiology of the organisms (Adams et al., 2007 ▸; Desler et al., 2009 ▸).

An earlier study identified B. pseudomallei genes that encode proteins which are crucial for survival and adaptation of the bacterium in different environmental niches (Wong et al., 2022 ▸). One of the identified genes was bpsl0741, which is annotated as encoding a hypothetical protein (HP). In an attempt to determine the function of this proposed essential protein, we overexpressed, purified, characterized, crystallized and performed an initial X-ray crystallographic analysis of recombinant BPSL0741 (rBPSL0741). Based on the Burk­holderia Genome Database (https://www.burkholderia.com/), BPSL0741 has 185 amino acids and the encoding gene bpsl0741 lies in chromosome 1 (https://www.ncbi.nlm.nih.gov/protein/CAH34733.1). Sequence analysis revealed that BPSL0741 belongs to protein family PF08937 with a domain of unknown function, DUF1863 (https://www.burkholderia.com/feature/show/?id=370459&view=functions). The BPSL0741 sequence shares 29.8% sequence identity (with 85% query coverage) with the sequence of a putative signal transduction protein from Agathobacter rectalis ATCC 33656 (PDB entry 3hyn; Joint Center for Structural Genomics, unpublished work). Upon further examination, the function of this protein (PDB entry 3hyn) has remained uncharacterized. InterPro predicted that BPSL0741 contains a Thoeris protein ThsB TIR-like domain in the region of amino acids 24–120 (https://www.ebi.ac.uk/interpro/protein/UniProt/Q63WZ9/). Genome analysis revealed that the operon bpsl0741–bpsl0744 is a novel Indel that may function as a phage-related integrase (Sim et al., 2008 ▸; Supplementary Fig. S1).

2. Materials and methods

2.1. Protein expression and purification

The bpsl0741 gene was chemically synthesized using the gene-synthesis service provided by GenScript, USA. The synthetic fragment was cloned into the multiple cloning site of the pET-28b(+) plasmid flanked by NdeI and HindIII restriction sites. This produced a construct of the bpsl0741 gene fused to an N-terminal 6×His tag (Table 1 ▸). The pET-28b(+)-bpsl0741 construct was transformed into the expression host Escherichia coli BL21(DE3)pLysS by heat shock. The transformed bacterial cells were added to Luria–Bertani (LB) medium containing chloramphenicol (35 µg ml⁻¹) and kanamycin (35 µg ml⁻¹). The cells were then allowed to grow overnight at 310 K. The overnight bacterial cultures were subsequently transferred into 2 l LB medium and growth was continued until an OD₆₀₀ of 0.6 was achieved at 310 K. Next, 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to induce recombinant protein expression at 289 K for approximately 16 h. The bacterial cells were centrifuged at 10 000g followed by resuspension of the cell paste in binding buffer (25 mM Tris–HCl pH 7.5, 5 mM β-mercaptoethanol, 250 mM NaCl, 20 mM imidazole). The cells were subsequently subjected to sonication (amplitude 35%, 6 min; Qsonica; 10 s pulse on and 20 s pulse off) followed by centrifugation at 10 000g to obtain soluble protein. The supernatant containing soluble protein was loaded onto an Ni–NTA-coupled HisTrap HP 5 ml column (GE Healthcare, UK) that had been pre-equilibrated with 25 ml binding buffer. The recombinant BPSL0741 (rBPSL0741) protein was eluted using a linear gradient of elution buffer (25 mM Tris–HCl pH 7.5, 5 mM β-mercaptoethanol, 500 mM NaCl, 0.5 M imidazole).

Table 1. Macromolecule-production information.

Source organism	Burkholderia pseudomallei
DNA source	Chemically synthesized (GenScript, USA)
Expression vector	pET-28b(+)
Expression host	E. coli BL21(DE3)pLysS
Complete amino-acid sequence of the construct produced†	MGSSHHHHHHSSGLVPRGSHMAYRNGTYVAFHANGTNRPGGNSDIDYYNLLKAWVGKDDDHFTMNNSHDKASAVRDSSKKETLRASLLERLRNSKNMVLIIGDTTLLDDDWVPFEISSAVDTYEIPIIAVYTQYSTPIRNPAALRSRWPDALATRIDSQKAKVIHIPFKKAALNDAISQFSHNNMPKGPLSFYSDEAYESFGIDG

^†The His-tag fusion sequence at the N-terminus of rBPSL0741 is shown in bold, followed by the 185 amino-acid residues of rBPSL0741.

The rBPSL0741 protein eluted at 278 mM imidazole and the purified protein fractions were pooled and concentrated using a 3 kDa molecular-weight cutoff filter (Sartorius, Germany). The concentrated rBPSL0741 protein was further purified by size-exclusion chromatography using a HiLoad 16/600 Superdex 200 pg column (GE Healthcare, USA) pre-equilibrated with 25 mM Tris–HCl pH 7.5, 1.25 M NaCl, 5 mM β-mercaptoethanol. SDS–PAGE was used to verify the purity of the purified rBPSL0741 protein. The protein was stained with Coomassie Brilliant Blue R-250 staining solution. Protein concentration was assessed utilizing the Bradford assay (Bio-Rad, USA). The rBPSL0741 protein was buffer-exchanged into 25 mM Tris–HCl pH 7.5, 100 mM NaCl, 5 mM β-mercaptoethanol before crystallization. Size-exclusion chromatography (SEC) was also used to analyze the rBPSL0741 protein on a HiLoad 16/600 Superdex 75 pg column (GE Healthcare, USA) that had been pre-equilibrated with 25 mM Tris–HCl pH 7.5, 5 mM β-mercaptoethanol, 250 mM NaCl.

2.2. Mass-spectrometric analysis

Mass-spectrometric analysis was conducted in the Proteomics and Metabolomics (PROMET) Laboratory, Malaysian Palm Oil Board (MPOB). The SEC-purified rBPSL0741 (25 µg) was subjected to trypsin digestion according to the method of Lau & Othman (2019 ▸). The freeze-dried trypsin-digested peptides were resuspended in 25 µl 0.1% formic acid and 5% acetonitrile before liquid chromatography–mass spectrometry analysis using an EASY-nLC 1200 System (Thermo Scientific, Massachusetts, USA) as described by Lau et al. (2020 ▸). The Mascot sequence-matching software (Matrix Science) and the Ludwig NR Database (https://ludwig.guru/s/proteins+in+the+Nr+database) were used to confirm the identity of the rBPSL0741 protein.

2.3. Protein crystallization

The purified rBPSL0741 protein in buffer consisting of 25 mM Tris–HCl pH 7.5, 100 mM NaCl, 5 mM β-mercapto­ethanol was concentrated to 7.5 mg ml⁻¹ using a Vivaspin 2 polyethersulfone (PES) concentrator fitted with a 3 kDa molecular-weight cutoff filter (Sartorius, Germany). Initial crystallization screening was performed by sitting-drop vapor diffusion in 96-well MRC crystallization plates (Molecular Dimensions, USA) using the Crystal Screen 1 & 2 crystallization screening kits (Hampton Research, USA). Drops consisting of equal volumes of purified rBPSL0741 and reservoir solution (1 µl each) were equilibrated against 70 µl reservoir solution at 293 K. Initial crystal hits were observed after ∼72 h of incubation. The crystallization conditions were further optimized by switching to the hanging-drop vapor-diffusion method in 24-well plates at 293 K. A reservoir consisting of 0.1 M sodium acetate trihydrate pH 4.6, 0.15 M ammonium acetate, 30% PEG 4000 yielded crystals that were suitable for diffraction analysis after 14 days of incubation.

2.4. Data collection and processing

The rBPSL0741 crystals were soaked in a cryoprotectant consisting of protein buffer and reservoir solution [25 mM Tris–HCl pH 7.5, 100 mM NaCl, 5 mM β-mercaptoethanol, 0.1 M sodium acetate trihydrate pH 4.6, 0.15 M ammonium acetate, 32% PEG 4000 (representing ∼1.1 times the precipitant concentration of the crystal reservoir)] with an additional 10% glycerol for ∼5 min at 298 K. Diffraction data were acquired at 100 K in a nitrogen-gas stream using an in-house Rigaku MicroMax-007 HF X-ray diffractometer (Rigaku, Tokyo, Japan) at the Malaysia Genome and Vaccine Institute (MGVI). A total of 792 images with a 0.25° rotation range were collected with a PILATUS 200K detector. Several programs from the CCP4 software suite (Agirre et al., 2023 ▸) were used to process the X-ray diffraction data. The iMosflm interface (version 7.1.1; Battye et al., 2011 ▸) was used to index and integrate the data. The crystal lattice was analyzed using POINTLESS (Evans, 2006 ▸) and the scaled data were integrated with AIMLESS (Evans & Murshudov, 2013 ▸).

3. Results and discussion

rBPSL0741 was successfully overexpressed at 289 K as soluble protein, which was purified by affinity chromatography and SEC. From the SDS–PAGE profile, Ni–NTA purification resulted in a dominant protein band at ∼23 kDa, which corresponds to the calculated molecular weight of rBPSL0741 [∼22.9 kDa; calculated using ProtParam (Gasteiger et al., 2005 ▸) for BPSL0741 fused to an additional 20 amino acids of the 6×His tag at the N-terminus (Table 1 ▸)] that eluted at 278 mM imidazole (Fig. 1 ▸a, Supplementary Fig. S2).

Figure 1.

Overexpression and size estimation of the purified rBPSL0741 protein. (a) The rBPSL0741 protein band corresponded to a size between the 20 and 25 kDa protein markers on 12% SDS–PAGE. Lane M, BLUeye Prestained Protein Ladder (labeled in kDa). Lane 1, total protein of crude extract without IPTG induction (negative control); lane 2, total protein of crude extract after 1 mM IPTG induction; lane 3, supernatant of crude extract after IPTG induction; lane 4, pellet of crude extract after IPTG induction; lane 5, flowthrough of Ni²⁺–NTA affinity column during sample application; lane 6, eluate from Ni²⁺–NTA affinity-column purification of rBPSL0741 using a 5 ml HisTrap HP column (GE Healthcare, UK); lane 7, size-exclusion chromatography of purified rBPSL0741 using a HiLoad 16/600 Superdex 200 pg gel-filtration column (GE Healthcare, UK). (b) Size-exclusion chromatogram of rBPSL0741 using a HiLoad 16/600 Superdex 200 pg gel-filtration column pre-equilibrated in 25 mM Tris–HCl pH 7.5, 1.25 M NaCl, 5 mM β-mercaptoethanol. rBPSL0741 eluted at a retention volume of ∼78 ml.

The Ni–NTA-purified protein was further purified to homogeneity using SEC (Fig. 1 ▸b). During SEC purification, a buffer with a high salt concentration (1.25 M) was applied as it enabled better protein recovery. Overall, the two-step purification effectively produces adequate amounts of rBPSL0741 protein at high purity, as shown by SDS–PAGE (lane 7, Fig. 1 ▸a). The oligomerization of rBPSL0741 was further estimated using SEC. The SEC results indicated that the eluted recombinant protein is likely to be monomeric, with retention volumes that correspond to an ∼18 kDa protein, i.e. smaller compared with the estimated size of ∼23 kDa for rBPSL0741 (Supplementary Figs. S2b and S2c). Nonetheless, it has been well documented that protein characteristics such as the number of charged groups, the hydrophobicity and the molecular shape may also influence the SEC separation (Hong et al., 2012 ▸). Mass-spectrometric analysis confirmed the SEC-purified recombinant protein to be rBPSL0741. A total of 17 peptides with sequences covering 60% of the rBPSL0741 amino-acid content were identified (Supplementary Fig. S1).

The validated SEC-purified rBPSL0741 (7.5 mg ml⁻¹) was subjected to crystallization screening. The first crystals were obtained using a reservoir solution consisting of 0.1 M sodium acetate trihydrate pH 4.6, 0.2 M ammonium acetate, 30% PEG 4000 (condition A10 of Crystal Screen 1 & 2; Hampton Research, USA) after ∼72 h. The protein crystal appeared as a cluster of long and thin crystal rods. Optimization of the initial hit conditions yielded rBPSL0741 protein crystals that were appropriate for X-ray diffraction using 0.1 M sodium acetate trihydrate pH 4.6, 0.15 M ammonium acetate, 32% PEG 4000 (Table 2 ▸). The crystals grew to ∼300 × 75 × 50 µm after 14 days (Fig. 2 ▸a).

Table 2. Crystallization information.

Method	Sitting-drop vapor diffusion for initial crystal screening, hanging-drop vapor diffusion for crystal optimization
Plate type	96-well MRC plates for initial crystal screening and 24-well plates for crystal optimization
Temperature (K)	293
Protein concentration (mg ml−1)	7.5
Protein buffer composition	25 mM Tris–HCl pH 7.5, 100 mM sodium chloride, 5 mM β-mercaptoethanol
Composition of reservoir solution	0.1 M sodium acetate trihydrate pH 4.6, 0.15 M ammonium acetate, 30% PEG 4000
Volume and ratio drop	2 µl; 1:1 protein:reservoir solution
Volume of reservoir solution	70 µl for initial crystal screening, 1.0 ml for optimization

Figure 2.

The rBPSL0741 protein crystal and its X-ray diffraction image and analysis. (a) The rBPSL0741 crystals were grown using reservoir solution consisting of 0.1 M sodium acetate trihydrate pH 4.6, 0.15 M ammonium acetate, 30% PEG 4000. (b) The diffraction image of rBPSL0741 extends to a resolution of ∼2.0 Å as viewed by the DIALS image viewer in Phenix 1.21-5207 (Liebschner et al., 2019 ▸). (c) The self-rotation function of the BPSL0741 data (45–3.5 Å) presented as a stereographic projection calculated by MOLREP (Vagin & Teplyakov, 2010 ▸). Three perpendicular twofold crystallographic axes of point group 222 are presented in the χ = 180° section. The x, y and z axes of the plot align with the a, b and c crystallographic axes, respectively.

The protein crystals were flash-cooled and diffracted to 2.1 Å resolution using the in-house X-ray source (Fig. 2 ▸b). The data were collected with 99.2% completeness and were subjected to processing and indexing with iMosflm (version 7.1.1), after which AIMLESS was used to scale and merge the data. The crystal lattice was shown to be orthorhombic, with POINTLESS (Evans, 2006 ▸) indicating that the crystal belonged to space group P2₁2₁2₁. Data-collection and processing statistics for the scaled and integrated data from AIMLESS (Evans & Murshudov, 2013 ▸) are summarized in Table 3 ▸. A self-rotation function calculation for the rBPSL0741 X-ray diffraction data using MOLREP (Vagin & Teplyakov, 2010 ▸) indicated three perpendicular twofold crystallographic axes of point group 222 in the κ = 180° section of the self-rotation function map (Fig. 2 ▸c). Nonetheless, the possible Matthews coefficient (V_M) was calculated to be approximately 2.18 ų Da⁻¹, suggesting that the crystal contained 43.49% solvent with two molecules in the asymmetric unit. While the SEC results indicate that rBPSL0741 is likely to be a monomeric protein, it is important to solve the crystal structure for a better understanding of the two molecules in the asymmetric unit. The parameters and data-collection statistics of the crystal are shown in Table 3 ▸.

Table 3. Data collection and processing.

Values in parentheses are for the outer shell.

Diffraction source	Rigaku X-ray diffractometer
Wavelength (Å)	1.54187
Temperature (K)	100
Total No. of reflections	71232
No. of unique reflections	21836
Space group	P212121
a, b, c (Å)	62.92, 64.57, 89.16
α, β, γ (°)	90.00, 90.00, 90.00
Mosaicity (°)	0.57
Resolution range (Å)	45.06–2.09 (2.15–2.09)
Completeness (%)	99.2 (94.3)
Multiplicity	3.3 (2.4)
Mean I/σ(I)	4.3 (2.1)
R meas †	0.201 (0.488)
CC1/2	0.978 (0.838)
Overall B factor from Wilson plot (Å2)	4.7

^†R_meas = , where N(hkl) is the multiplicity of reflection hkl.

As we are still in the process of determining the structure of rBPSL0741, in silico structure prediction was conducted for BPSL0741 with AlphaFold2 using the ColabFold server (https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb). The best AlphaFold2-predicted structure was shown to have low confidence (70 > pLDDT > 50) in the N-terminus (residues 1–70) and loop region (residues 81–92), but confidence (90 > pLDDT > 70) or high confidence (pLDDT > 90) in the C-terminal region (residues 73–183, except for the loop region 81–92) (Supplementary Fig. S3). In addition, the prediction aligned error (PAE) score was also relatively low for the structure at the N-terminus. Since only half of the BPSL0741 protein structure could be predicted with high confidence, it is crucial to determine the experimental crystal structure, which would assist in unraveling the biological function of BPSL0741, which has been predicted to contain a TIR-like domain.

4. Conclusion

The rBPSL0741 protein predicted to contain a virulence-associated TIR-like domain was successfully overexpressed in soluble form, purified to homogeneity and crystallized. rBPSL0741 is likely to be a monomeric protein in solution. We are working towards the structure determination of rBPSL0741 by molecular replacement. We aim to reveal the function of BPLS0741 by comparing the structure of rBPSL0741 with the structures of the putative signal transduction protein (PDB entry 3hyn) from A. rectalis and the Thoeris protein ThsB TIR-like domain, followed by experimental validation of the function of BPSL0741, which may be associated with adaptation and survival of B. pseudomallei in the infected host.

5. Related literature

The following reference is cited in the supporting information for this article: Kelley et al. (2015 ▸).

Funding Statement

This work was funded by Ministry of Higher Education, Malaysia grant FRGS/1/2018/STG04/UKM/02/3.