Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

1. Preparing Chl a Stock Solutions

1. CRITICAL STEP: Perform all the steps of chlorophyll preparation in a chemical hood, under green light (520 nm) or in the dark in order to minimize photodamage. Always add Nitrogen or Argon before freezing the pigments for storage. Ensure that all solvents are analytical grade.
2. Weigh about 5 mg of lyophilized Spirulina platensis cells or other cyanobacterium cells containing only Chl a in thylakoid membranes and crush it using a mortar and pestle.
3. Load crushed cells onto a glass column and wash with about 50-100 ml of 100% acetone in order to remove carotenoids. Discard the eluted orange/green fraction.
   Note: If the orange fraction is not eluted with 100 ml of acetone continue washing the cells with acetone until green fraction starts to elute.
4. Exchange acetone with 100% methanol and collect the green fraction containing Chl a. The volume of eluted fraction may vary between 50-100 ml. At the beginning, the eluted fraction has a dark green color, which changes into pale green at the end of elution. When the color of eluted fraction turns into pale green stop the elution.
5. Evaporate the methanol using a rotary evaporator until the extract is completely dry. Do not apply heat to the solution; ensure that the evaporator's water bath temperature does not exceed 30 °C.
   Note: The time of evaporation depends on the volume of methanol fraction being evaporated and may vary between 10-60 min. It is important to dry the extract completely.
6. Dissolve the pigments from the dried extract in a small volume of diethyl ether (about 5-10 ml), and filter through cotton wool. Ensure that pigments are completely dissolved in ether before filtering.
7. Evaporate the diethyl ether until the pigments are completely dry (10-30 min).
   Note: The dry pigments can be purged with and kept under Nitrogen or Argon at -20 °C, in the dark until further processing.
8. Dissolve the dry pigments in the smallest volume possible of 100% methanol (about 1 ml), even if not everything is completely suspended. Add 4 ml of acetone to the solution, and flick the glass gently in order to completely dissolve the pigments.
9. Using a Pasteur pipette, load the sample gently onto a column of DEAE sepharose equilibrated in 100% acetone.
10. Elute carotenoids (an orange-yellow band) with 100% acetone. Then, elute Chl a (green band) with 3:1 v/v acetone/methanol mixture.
    Note: The volume of acetone and acetone/methanol mixture is approximately equivalent to the volume of DEAE sepharose loaded on the column.
11. Verify Chl a purity by thin layer chromatography using a 68:25:5:2 dichloromethane/n-hexane /isopropanol /methanol (v/v) mixture as eluent ¹⁷.
12. Evaporate the solvent using a rotary evaporator until the Chl a is completely dry (10-60 min).
    Note: The dry Chl a can be purged with nitrogen or argon and stored under argon atmosphere at -20 °C in the dark.
13. Prepare Chl a stock solution by re-dissolving the dry Chl a in 2-4 ml of 100% ethanol.
    Note: The extinction coefficient of Chl a at 663 nm is 74,400 cm^-1M^-1 (83.3 cm^-1 (mg/ml)^-1) in ethanol. A typical stock solution should have an OD of 1,860 corresponding to a concentration of 25 mM (23 mg/ml). Adding 20 µl of this stock to an emulsion containing 5 ml of organic phase, and 1 mg of WSCP in 1 ml of aqueous phase results in a mixture with 10 fold molar excess of Chl a vs. WSCP.

2. Preparing Organic Phase of the Emulsion

Note: The organic phase of the emulsion is composed of mineral oil containing 4.5% (v/v) Span 80, and 0.4% (v/v) Tween 80.

1. Weigh in a 50 ml tube 0.2 g of Tween 80, 1.8 g of Span 80, and 38 g of mineral oil. Mix well all components and cool down on ice.
   Note: The organic phase can be stored in 4 °C up to one week.

3. Preparing the Aqueous Phase of the Emulsion

Note: The aqueous phase of the emulsion may be composed of either purified WSCP, or crude extract of bacteria overexpressing WSCP.

1. Preparing an aqueous phase containing purified WSCP.
   1. Grow E.coli BL21 bacteria containing WSCP plasmid ¹² in 1 L of LB medium at 37 °C until OD of 0.3-0.6.
   2. Induce protein expression by adding 1 mM IPTG. After induction grow bacteria at 30 °C for 12-16 hr.
   3. Harvest bacteria by centrifugation at 5,000 x g for 10 min at 4 °C.
   4. Dissolve the pellet in binding buffer and sonicate on ice (30 sec on, 15 sec off, five times). Use 10 ml of buffer for the pellet obtained from centrifugation of 250 ml of LB medium with cells overexpressing the protein.
      Note: Depending on purification method, the binding buffer may be composed of 100 mM phosphate buffer, pH 7.2 or 50 mM Tris, pH 7.5, 500 mM NaCl, 5 mM EDTA.
   5. Spin down the cell lysate at 12,000 x g for 30 min at 4 °C.
   6. Purify WSCP by affinity chromatography. Depending on the tag fused to WSCP, purify recombinant proteins using the appropriate commercially available affinity chromatography system for protein purification following manufacturer instructions.
   7. For emulsion preparation, use purified WSCP in 50 mM phosphate buffer, pH 7.8. Ensure that the final protein amount used for reconstitution is 0.5-1.0 mg per 1 ml of buffer.
2. Preparing an aqueous phase containing crude bacterial lysate with WSCP.
   1. Grow E.coli BL21 cells containing plasmid-expressing WSCP in 250 ml of LB medium at 37 °C until OD 0.3-0.6.
   2. Induce protein expression with 1 mM IPTG and grow bacteria overnight at 30 °C.
   3. Harvest bacterial cells by centrifugation at 5,000 x g for 10 min at 4 °C.
   4. Dissolve the pellet in 1-2 ml of 50 mM sodium phosphate buffer pH 7.8, sonicate (30 sec on, 15 sec off, three times) and centrifuge at 12,000 x g for 30 min at 4 °C.
   5. Prepare the aqueous phase of the emulsion by mixing 0.125 ml of supernatant with 0.875 ml of 50 mM sodium phosphate buffer pH 7.8.

4. Assembly of WSCP with Chl a in Emulsion

1. Transfer 5 ml of oil-surfactant mixture into a glass vial and cool it on ice. Before pipetting, verify that all the components of the organic phase are mixed thoroughly and there is no phase separation between surfactants and mineral oil.
2. Add 1 ml of ice-cold aqueous phase prepared as in section 3 to 5 ml of organic phase.
3. Mix both phases using a tissue homogenizer for 2 min at 9,500 rpm on ice.
4. CRITICAL STEP: From this stage on, perform all further steps under green light (520 nm) in order to minimize photodamage. Add 20 µl of 25 mM Chl a stock solution (see section 1.13) to the emulsion. Disperse by flicking and inverting the glass vial. Make sure that the Chl is evenly distributed in the emulsion.
5. Incubate the emulsion for 1-2 hr on ice in the dark.
6. In order to break down the emulsion and separate water droplets from the organic phase transfer the emulsion to 1.5 ml plastic tubes and centrifuge at 14,000 x g for 5 min at room temperature.
   Note: If the assembly is successful, the lower aqueous phase should have a green color.
7. Dispose the upper oil phase and add 1 ml of mineral oil. Mix well the mineral oil with the pelleted emulsion by vortex or by flipping the tube thoroughly. Spin down the sample at 14,000 x g for 5 min at room temperature. Repeat this step until a clear meniscus separating the aqueous and mineral oil phases, without any intermediate emulsion is obtained.
8. Perform this step in a chemical hood. After the aqueous and mineral oil phases are clearly separated, remove mineral oil and add 1 ml of water-saturated diethyl ether. Vortex and spin down the sample at 14,000 x g for 5 min at room temperature. Repeat this step twice.
9. After the second centrifugation, remove the diethyl ether and leave the tubes open for 5-20 min in the hood.
10. Finally, load the aqueous phase containing the WSCP/Chl a complex onto a desalting column and elute with buffer appropriate for further experiments.
    Note: The protein is stable in phosphate- and Tris buffers in a broad range of pH (6.0-7.5). The sample can be stored at 4 °C protected from light up to one month.