Isolation of magnetosomes

M. gryphiswaldense cells (approximately 10 g of wet weight) was suspended in a 100-ml buffer containing 20 mM of HEPES, 4 mM of EDTA, 0.1 mM of phenylmethylsulfonyl fluoride, pH 7.4 and disrupted three times using an ultra homogenizer (GEA Niro Soavi, Germany). The suspension was then centrifuged at 680 ×g for 10 minutes to remove the unbroken cells, and the cell debris and the supernatant were passed through a MACS magnetic separation column (Germany’s Miltenyi Biotec). To bind the magnetic particles to the column matrix, the columns were placed between two Sa-Co-magnets generating a magnetic field gradient inside the column. The separation of magnetosome particles was completed at the time when there was no trace of black magnetosome-like particles in the cell extract after passage through the column. The attached magnetic particles to the column were rinsed with 50 ml of 10 mM HEPES, 200 mM of NaCl, pH 7.4 and subsequently with 100 ml of 10 mM HEPES, pH 7.4, to eliminate electrostatically bound contamination. The column was then removed from the magnets, and magnetic particles were eluted from the column by flushing out with a 10-mM HEPES buffer. The magnetosome suspension was finally loaded on the top of a sucrose cushion (55% [wt/wt] sucrose in 10 mM HEPES, pH 7.4) and centrifuged in a swinging 50-ml bucket rotor at 280,000 ×g at 4 °C for 8 hours using an ultracentrifuge. The supernatant containing sucrose ingredient was removed, and the pelleted magnetosomes were suspended in 0.1-M PBS buffer (pH 7.4) and centrifuged at 11,000 ×g at 4°C for 8 minutes, finally disrupted using an ultrasonic cell crusher (300 W) for 4 s with an interval of 8 s and repeated 80 times. Extracted magnetosomes were then absorbed to the bottom of the beaker using a magnetic field, and the cell debris was removed by discarding the supernatant. The magnetosomes were resuspended in 0.1 M of PBS (pH 7.4) and ultrasonically cleaned (45 W, for 4 s with an interval of 8 s and repeated 40 times) and collected. All the processes were repeated 15 times. The purified magnetosomes were finally dried using a vacuum freeze-drying method (Kinetics, EZ550Q) and kept at 105 °C for 24 h and weighed. The purified magnetosomes were sterilized by Co60 irradiation (15 kGy) and resuspended in 0.1 M of PBS (pH 7.4) at a concentration of 500 μg/mL[26].