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Updated: Jun 13, 2025
Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG
Published on: May 16, 2017
Protein expression, purification, crystallization and crystallographic studies of BPSL0741 from Burkholderia
Nurul Fadzillah Fadhar1, Pravin Kumran Nyanasegran1, Mohd Firdaus-Raih1
1Institute of Systems Biology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia.
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 (VM) was calculated to be 2.18 Å3 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.
Insights
Researchers crystallized a hypothetical protein, BPSL0741, from Burkholderia pseudomallei, the bacterium causing melioidosis. This structural study is crucial for understanding bacterial survival mechanisms and developing new treatments against antibiotic-resistant strains.
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- * Burkholderia pseudomallei causes melioidosis, a severe disease with increasing antibiotic resistance.
- * Identifying bacterial survival genes is key to developing new therapeutic strategies.
- * The gene bpsl0741 encodes a hypothetical protein potentially involved in bacterial host survival.
Purpose of the Study:
- * To express, purify, and crystallize the hypothetical BPSL0741 protein.
- * To determine the preliminary crystallographic data of BPSL0741.
- * To lay the groundwork for future structural and functional analyses of BPSL0741.
Main Methods:
- * Recombinant expression and purification of BPSL0741 protein.
- * Mass spectrometry for protein verification.
- * X-ray diffraction analysis of crystallized BPSL0741.
Main Results:
- * Recombinant BPSL0741 (rBPSL0741) was successfully expressed and purified.
- * Crystals of rBPSL0741 were obtained using vapor diffusion.
- * The crystals diffracted to 2.1 Å resolution in an orthorhombic space group.
Conclusions:
- * The BPSL0741 protein has been successfully crystallized, enabling further structural studies.
- * The obtained crystallographic data suggest suitability for detailed structural determination.
- * Understanding BPSL0741 structure may reveal novel targets for combating melioidosis.
