Expression, purification, crystallization and preliminary X-ray crystallographic studies of a mitochondrial membrane-associated protein Cbs2 from Saccharomyces cerevisiae

Abstract

Background: Mitochondria are unique organelles that are found in most eukaryotic cells. The main role of the mitochondria is to produce ATP. The nuclear genome encoded proteins Cbs1 and Cbs2 are located at the mitochondrial inner membrane and are reported to be essential for the translation of mitochondrial cytochrome b mRNA. Genetic studies show that Cbs2 protein recognizes the 5' untranslated leader sequence of mitochondrial cytochrome b mRNA. However, due to a lack of biochemical and structural information, this biological process remains unclear. To investigate the structural characteristics of how Saccharomyces cerevisiae (S. cerevisiae) Cbs2 tethers cytochrome b mRNA to the mitochondrial inner membrane, a preliminary X-ray crystallographic study was carried out and is reported here.

Methods: The target gene from S. cerevisiae was amplified by polymerase chain reaction. The PCR fragment was digested by the NdeI and XhoI restriction endonucleases and then inserted into expression vector p28. After sequencing, the plasmid was transformed into Escherichia coli C43 competent cells. The selenomethionine derivative Cbs2 protein was overexpressed using M9 medium based on a methionine-biosynthesis inhibition method. The protein was first purified to Ni²⁺-nitrilotriacetate affinity chromatography and then further purified by Ion exchange chromatography and Gel-filtration chromatography. The purified Se-Cbs2 protein was concentrated to 10 mg/mL. The crystallization trials were performed using the sitting-drop vapor diffusion method at 16 °C. The complete diffraction data was processed and scaled with the HKL2000 package and programs in the CCP4 package, respectively.

Results: Cbs2 from S. cerevisiae was cloned, prokaryotic expressed and purified. The analysis of the size exclusion chromatography showed that the Cbs2 protein peaked at a molecular weight of approximately 90 KDa. The crystal belonged to the space group C2, with unit-cell parameters of a = 255.11, b = 58.10, c = 76.37, and β = 95.35°. X-ray diffraction data was collected at a resolution of 2.7 Å. The Matthews coefficient and the solvent content were estimated to be 3.22 Å 3 Da-1 and 61.82%, respectively.

Conclusions: In the present study Cbs2 from S. cerevisiae was cloned, expressed, purified, and crystallized for structural studies. The molecular weight determination results indicated that the biological assembly of Cbs2 may be a dimer.The preliminary X-ray crystallographic studies indicated the presence of two Cbs2 molecules in the asymmetric unit. This study will provide an experimental basis for exploring how Cbs2 protein mediates cytochrome b synthesis.

Keywords: Cbs2; Crystallization; Cytochrome b; Expression and purification; Mitochondria.

Figure 1. The Cbs2 expression plasmid and the encoded protein.

(A) The 6-His tag and the cleavage site are marked with black lines. (B) Sequence of Saccharomyces cerevisiae Cbs2 protein. The methionines are labelled in red.

Figure 2. Purification of the Cbs2 protein by Ni NTA affinity chromatography and FPLC Resource Q anion-exchange chromatography (GE Healthcare).

(A) SDS-PAGE (12%) analysis of Ni NTA affinity purified Cbs2 protein. L, Ladder; C,Crude; S, Supernatant; R1, Resin washed by 20 mL washing buffer containing 50 mM imidazole; R2, Resin washed by 20 mL washing buffer containing 70 mM imidazole; E, Elution of Cbs2; ER, Resin after elution. (B) Western blot analysis of Ni NTA affinity purified Cbs2 protein. L, Ladder; Cbs2-His tag, Purified Cbs2 protein with His tag. The identity of Cbs2 was confirmed by western blot, and as expected, the Cbs2 protein was recognized by mouse anti-His6 antibody IgG, followed by HRP-conjugated goat anti-mouse IgG Ab. (C) Hitrap Q anion-exchange chromatography profile of Cbs2 protein. Peak Cbs2 38.66mS/cm represents that Cbs2 protein was eluted when the conductance is 38.66 mS/cm. The green line, the gradient of the concentration of NaCl increased from 0.05M to 1 M. (D) SDS-PAGE (12%) analysis of Cbs2 protein purified by Hitrap Q. L, Ladder; C1–C7, fractions C1–C7 of Peak Cbs2.

Figure 3. Further purification of the Cbs2 protein by Superdex 200 10/300 gel-filtration chromatography colum (GE Healthcare).

(A) Size-exclusion chromatography profile of Cbs2 protein . Peak Cbs2 represents further purified Cbs2 protein. (B) SDS-PAGE (12%) analysis of Cbs2 protein purified by Superdex 200 10/300. L, Ladder; C1–C6, fractions C1–C6 of Peak Cbs2. (C) Standard chromatography profile determined by Gel Filtration Calibration Kit The protein standard peaks are: 1, thyroglobulin (669 kDa); 2, aldolase (158 kDa); 3, conalbumin (75 kDa); 4, ovalbumin (44 kDa) and 5, Ribonuclease A(13.7 kDa). B, Blue dextran (2,000 kDa) was used to determine the void volume (Vo) of the column. (D) The calibration curve is shown on the right side. Partition coefficient (Kav) was calculated from the formula, Kav = (VE−V0)/(VT−V0), where VE is the retention volume of each sample, VT is the total column volume, and V0 is the void volume, respectively. Kav was plotted against the molecular weight of proteins.

Figure 4. Crystallization and diffraction Data Collection of the recombinant Cbs2.

(A) A diffraction image of Cbs2 protein collected on beamline 17U1 at the Shanghai Synchrotron Radiation Facility (SSRF) at 100 K. (B) Measurability of anomalous signal. (C) Electron density map of Cbs2. (D) Cartoon representation of Cbs2.