The overall goal of this technique is to enable incorporation of water-insoluble chlorophylls into the binding sites of water-soluble proteins. This method enables assembly of chlorophylls with recombinant proteins, which allows rigorous studies of chlorophyll-protein interactions and opens up new possibilities for constructing novel chlorophyll-protein complexes. The main advantage of this technique is that it does not require tagging or immobilizing the proteins.
Thus, it is well-suited for screening assays. Demonstrating the procedure will be Dominica Bednarczyk, a post-doc in my research group. While preparing the following stock solutions perform all chlorophyll preparation steps under a chemical hood under green light or in the dark to minimize photo damage.
Always add nitrogen or argon before freezing the pigments for storage and ensure that all solvents are analytical grade. To begin, weigh about five grams of lyophilized Spirulina platensis cells or other cyanobacterium cells containing only chlorophyll a or chlorophyll-a in thylakoid membranes and use a mortar and pestle to crush it. The critical step during the chlorophyll extraction is to make sure that cyanobacteria cells are thoroughly lyophilized and to ensure the complete removal of water from the DEAE-Sepharose by several washes with acetone.
Load the crushed cells onto a glass column and with about 50-100 milliliters of 100%acetone wash the tissue to remove the carotenoids. After washing, discard the eluted orange-green fraction. The actual color may vary from orange-green to green depending on the cyanobacteria culture and growth conditions.
Exchange the acetone with 100%methanol and collect the green fraction containing chlorophyll a. When the color of the eluted fraction turns from dark green to pale green stop the elution. Next, using a rotary evaporator with a water bath that does not exceed 30 degrees Celsius evaporate the methanol until the extract is completely dry then use a small volume of diethyl ether to completely dissolve the pigments from the dried extracts and filter the solution through cotton wool, using additional small volume of ether to wash pigments off the cotton wool.
Evaporate the diethyl ether until the pigments are completely dry. If stopping at this point, keep the dry pigments under nitrogen or argon at 20 degrees Celsius in the dark until further processing. To proceed with chlorophyll a preparation partially dissolve the dry pigments in the smallest volume possible of 100%acetone, then add 4 milliliters of acetone to the suspension and swirl the flask to completely dissolve the pigments.
Using a Pasteur pipette gently load the sample onto a DEAE-Sepharose column equilibriated in 100%acetone, and with 100%acetone, elute the carotenoids. Then use a 3:1 volume-to-volume acetone-methanol mixture to elute the chlorophyll a. You may use a flashlight to briefly illuminate the column during elution in order to observe the actual pigment colors.
With thin-layer chromatography, verify chlorophyll a purity using a 68:25:5:2 volume-to-volume ratio of dichloromethane and hexane, isopropanol and methanol to elute the sample. Then, using a rotary evaporator, evaporate the solvent until the chlorophyll a is completely dry. To prepare a chlorophyll a stock solution re-dissolve the dry chlorophyll a in 2-4 milliliters of 100%methanol.
To prepare the organic phase of the emulsion, in a 50 milliliter tube weigh 0.2 grams of Tween 80, 1.8 grams of Span 80, and 38 grams of mineral oil. Mix the components well and place the solution on ice to cool. To prepare an aqueous phase containing purified water-soluble chlorophyll-binding protein, or WSCP, grow E.coli BL21 bacteria containing the WSCP gene in 1 liter of LB medium at 37 degrees Celsius to an OD600 of 0.3 to 0.6.
Add 1 millimolar IPTG to induce protein expression and grow the bacteria at 30 degrees Celsius for 12-16 hours, then harvest the bacteria by centrifugation at 5000 G and 4 degrees Celsius for 10 minutes. With a buffer suitable for the affinity chromatography dissolve the pellet, transfer the bacterial suspension to smaller tubes and sonicate on ice. After spinning down the lysate, purify the recombinant tagged WSCP proteins using the appropriate commercially-available affinity chromatography system for protein purification following the manufacturer's instructions.
Prepare the aqueous phase of the emulsion by exchanging the buffer to 50 millimolar phosphate buffer pH 7.8 and adjusting the purified WSCP concentration to 0.5 to 1 milligram per milliliter. To prepare an aqueous phase containing crude bacterial lysate with WSCP, grow E.coli BL21 cells containing a plasmid expressing WSCP in 250 milliliters of LB at 37 degrees Celsius to log phase. Add 1 millimolar IPTG to induce protein expression and grow the bacteria at 30 degrees Celsius overnight.
The following day, after harvesting the bacterial cells use 1-2 milliliters of 50 millimolar sodium phosphate buffer pH 7.8 to dissolve the pellet. Then, after sonicating, centrifuge the lysate at 12, 000 G and 4 degrees Celsius for 30 minutes. Prepare the aqueous phase of the emulsion by mixing 0.125 milliliters of supernatant with 0.875 milliliters of 50 millimolar sodium phosphate buffer pH 7.8.
To assemble WSCP with chlorophyll a in emulsion, transfer 5 milliliters of a thoroughly-mixed oil-surfactant mixture into a glass vial and cool on ice. Add 1 milliliter of ice-cold aqueous phase just prepared and use a tissue homogenizer at 9, 500 RPM on ice for two minutes to mix both phases. From this point forward, performing all steps under green light to minimize photodamage, add 20 microliters of 25 millimolar chlorophyll a stock solution to the emulsion.
Disperse by flicking and inverting the glass vial ensuring that the chlorophyll a is evenly distributed in the emulsion, then incubate on ice in the dark for 1-2 hours. The critical step during emulsion preparation is to cool down both the water phase and organic phase before mixing. Next, in order to break down the emulsion and separate the water droplets from the organic phase, transfer the emulsion to 1.5-milliliter plastic tubes and centrifuge at 14, 000 G at room temperature for five minutes.
Dispose of the upper oil phase and add 1 milliliter of mineral oil to the lower phase, then mix well by vortexing or by flipping the tube. After spinning the sample, remove the upper oil phase and repeat the addition of mineral oil and centrifugation until a clear meniscus separating the aqueous and mineral oil phases without an intermediate emulsion is achieved. Now, to the aqueous phase add 1 milliliter of water-saturated diethyl ether.
Then, after vortexing the sample, spin it down at 14, 000 G for 5 minutes at room temperature. After the third centrifugation, in the hood, remove the diethyl ether and leave the tubes open for 5-20 minutes. Finally, load the aqueous phase containing the WSCP-chlorophyll a complex onto a desalting column and elute with a buffer appropriate for future experiments.
Store the sample at 4 degrees Celsius away from light for up to one month. Using the protocol described in this video, recombinant WSCP apoproteins were assembled with chlorophyll a in water and oil emulsions. Shown here are the absorbances and CD spectra of chlorophyll a complexes with four recombinant WSCP variants.
The results are similar in band-shaped end position to previously reported spectra of the respective native complexes. This figure shows the positive assembly of WSCP with chlorophyll a in lysates from WSCP-expressing cells as seen by the green color of the aqueous phase. This result demonstrates the potential of water and oil emulsions as a fast screening system for positive assembly of WSCPs with chlorophylls.
As demonstrated here, droplets of WSCP-containing lysates feature distinct chlorophyll a fluorescence in confocal microscope images. This implies that successful assembly of WSCP-chlorophyll complexes can be detected directly in the water droplets and it may provide a basis for water-and-oil-emulsion-based screening systems. Once mastered, this technique can be done for five hours if it's performed properly.
While attempting this procedure, it is important to remember that working with chlorophylls should be done quickly, without exposing chlorophyll to light and oxygen. After its development, this technique enabled us to prepare a wide range of water-soluble chlorophyll-binding proteins that are used to explore the effect of protein environment on the electrolytic and spectral properties of chlorophylls. After watching this video, you should have a good understanding of how to extract and purify chlorophylls from cyanobacteria and assemble them in water-soluble proteins by using water and oil emulsions.
Don't forget that working with organic solvents, such as ether, methanol or acetone, can be harmful and precautions, such as working in a chemical hood, should always be taken while performing this procedure.
