Isolation and Characterization of Intact Phycobilisome in Cyanobacteria

The current protocol details the isolation of phycobilisomes from cyanobacteria by centrifugation through a discontinuous sucrose density gradient. The fractions of intact phycobilisomes are confirmed by 77K fluorescent emission spectrum and SDS-PAGE analysis. The resulting phycobilisome fractions are suitable for negative staining of TEM and mass spectrometry analysis.

Transcript

It is a simple and a fast protocol that allow people unfamiliar with cyanobacteria to isolate phycobilisome at ease. The advantage of this technique is that it is adapted to a small scale and it is easy to perform. It is a simple, reliable, and efficient method for isolating intact phycobilisomes of model and non-model cynobacteria.

Begin by transferring the cell culture of 0.8 OD at 750 nanometers into a one liter flask and dilute it in 500 milliliters of B-HEPES medium to a achieve the OD of 0.2 at 750 nanometers. Grow the culture with constant stirring at 30 degree Celsius until the OD reaches 0.8 to 1, indicating a late exponential growth phase. Centrifuge the culture at a speed of 10, 000 times g for 20 minutes at room temperature and discard the supernatant.

Suspend the cell pellets and washed twice with 0.75 molar potassium phosphate buffer, pH seven. Pellet the cells in a 50 milliliter centrifuge tube by centrification at room temperature. Discard the supernatant and resuspend the cells with 0.75 molar potassium phosphate buffer.

Measure the wet weight of the pellet by an electronic balance and resuspend the one gram wet weight of the pellet in five milliliters of the buffer. Add one milliliter of cell suspension and 0.4 to 0.6 grams of 0.1 millimeter glass beads into a two milliliter screw cap vial. Break the cells for 30 seconds by a bead beater.

Allow the tubes to cool in a room temperature water bath for two minutes. After lysis, centrifuge the vials for 10 seconds at room temperature at a speed of 500 times g. Collect the supernatant with a pipette into a 15 milliliter centrifuge tube.

Wash the beads with 0.5 milliliters of 0.75 molar potassium phosphate buffer one time, then transferred to the same centrifuge tube. Add Triton X-100 to the lysed cell suspension and incubate on a rocking shaker at room temperature and 40 RPM for 20 minutes or until the solution becomes homogenous. Centrifuge the tubes for 20 minutes at 17, 210 times g and store the supernatant without the upper Triton micelle layer at room temperature for up to one hour.

Make four concentrations of sucrose in 0.75 molar potassium phosphate buffer at pH seven. Place 2.8 milliliters of two molar sucrose buffer at the bottom of the 40 milliliter centrifuge tube and overlay with three layers of sucrose solutions. Finally, add three milliliters of PBS containing the supernatant fraction and centrifuge the resulting gradients.

Upon ultracentrification, condense the fractionated PBS and phycobiliproteins between the layers and observe the blue bands in Syn6803 and interfaces. Remove the sucrose by repeatedly concentrating and diluting the fractions with 0.75 molar potassium phosphate buffer and membrane the centrifugal filter units. For measuring PBS fluorescence dilute the concentrated PBS sample with 0.75 molar potassium phosphate buffer to make up to 500 microliters volume.

Add the diluted PBS sample to a transparent glass tube and freeze the tube in liquid nitrogen until it is entirely frozen. Move the frozen tube to a transparent dewar container pre-filled with liquid nitrogen. Complete cell lysis shows the supernatant and dark blue-green color before centrification.

The sucrose density gradient separation of isolated PBS shows several fractions of dissociated phycobiliproteins and the lowest fraction represents the intact PBS. The absorption spectra from the dissociated and intact PBS of JSC one and Syn6803 have the same peak at 622 nanometers, corresponding to the phycocyanin. Moreover, the phycoerythrin absorption peak at 571 nanometers in both dissociated and intact PBS of JSC-1.

The emission spectra of the dissociated PBS have one strong peak at approximately 650 nanometers in Syn6803 and JSC-1, representing the emission from phycocyanin. The fluorescent emission maximal peaks at 651 nanometers and 684 nanometers reveal that the energy harvested by phycocyanin was transferred efficiently to allophycocyanin and terminal emitters, which are ApcD and ApcE, in the core. The figure shows zinc stained SDS-PAGE under UV irradiation where the phycobiliprotein subunits are strongly fluorescent and the ApcE showed weak fluorescence, indicated with the arrow.

Kumasi blue staining shows that the upper fractions contain other non-PBS water soluble proteins and the intact PBS and Syn6803 shows a prominent ApcE band, indicated with an arrow, lacking in the dissociated PBS fractions. Ultracentrifugation demonstrates that the sucrose gradient prepared at a ratio of 1:3 displays wider bands than the gradient made at a ratio of 1:10. It is important to lysis the cells and solvolysis the supernatants completely to obtain more phycobilisomes.

Other methods, like cryo-EM or mixed spectrometry, can be used to study the protein structure or the protein composition of phycobilisomes.

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Journal (Biology)

Journal (Biology)

Isolation and Characterization of Intact Phycobilisome in Cyanobacteria

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