Extraction and purification of the photosynthetic ACIII from R. castenholzii

Purification of ACIII from Roseiflexus castenholzii

Through bioinformatics analysis of ACIII and modification of an existing isolation procedure (Reference 1, 2, 3), the critical step is the cation exchange chromatography that aims to obtain a purified preparation of the alternative complex III (ACIII) without the contamination of the LH-RC core complex of the thermophilic bacterium Roseiflexus castenholzii.

A suspension of Whole membranes (with OD₈₈₀ = 20 cm^-1) in 20 mM Tris-HCl pH = 8.0 (buffer A) was treated with 1% β-Octyl glucoside and stirred for 1hr at room temperature in the dark. The extraction was then centrifugation at 200,000 g for 2 hr (Ti 70 rotor, 45,000 rpm) at 4 ºC. The pellets were re-suspended in 50 mM sodium acetate pH = 5.0 (buffer B) and treated with 0.5% β-dodecyl maltoside as above with 1% β-Octyl glucoside. The supernatant from the second ultracentrifugation was collected and filtered through 0.22μm Millipore filter and subsequently loaded on a prepacked cation exchange chromatography column SPHP5 (GE Healthcare), which has been equilibrated with buffer B containing 0.04 % β-dodecyl maltoside (which makes up buffer C). The column was extensively washed with 50 mM NaCl in buffer C until the eluent is colorless. Finally, the crude ACIII was eluted from the column by a sodium gradient from 0.1 M NaCl to 0.4 M NaCl with 50 mL of buffer C at a flow rate of 2 mL/min. The collected fractions were concentrated and further purified by Superdex-200 gel filtration in buffer D (100 mM NaCl, 0.02% β-dodecyl maltoside, and 20 mM Tris-HCl, pH 8.0). The fractions with an absorption ratio of A₄₁₃/_A280 higher than 1.38 were pooled and used for cryo-EM. 

The polypeptide composition of the purified complex was determined by Tricine-SDS-PAGE and blue-native PAGE. The sample-solubilization condition was optimized by dissolving samples in buffer containing 5% 2-mercaptoethanol for 30 min at 65 °C, these conditions yielded the sharpest protein bands. The purified ACIII complex exists as a monomer with apparent molecular weight about 300 kD. The identity of SDS-PAGE and blue-native PAGE band was confirmed by MALDI-TOF mass spectroscopy. Each band was excised from the gel and analyzed by mass spectroscopy to show the exact subunit composition, the results showed that all of the components of ACIII were observed in our purified sample. 

References

1. M. F. Yanyushin, M. C. del Rosario, D. C. Brune, R. E. Blankenship, New class of bacterial membrane oxidoreductases. Biochemistry 2005, 44: 10037-10045.
2. X. Gao, Y. Xin, P. D. Bell, J. Wen, R. E. Blankenship, Structural analysis of alternative complex III in the photosynthetic electron transfer chain of Chloroflexus aurantiacus. Biochemistry 2010, 49: 6670-6679.
3. M. F. Yanyushin, Fractionation of cytochromes of phototrophically grown Chloroflexus aurantiacus. Is there a cytochrome bc complex among them? FEBS Lett. 512, 125–128 (2002).

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