In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

1. Total Pigment Extraction from Spinach Leaves

1. Homogenize one handful of spinach leaves (~20 g) in 100 ml of cold Grinding Buffer (see Table 1) using a blender for 20 sec.
2. Filter the solution through a two layers of nylon cloth with a pore diameter of 20 μm and centrifuge the filtrate at 1,500 x g for 10 min at 4 °C.
3. Resuspend the pellet containing the chloroplasts with a soft artists paint brush in 1 ml of cold Wash Buffer (see Table 1). Once the pellet is resuspended, add 50 ml of Wash Buffer and centrifuge the solution at 10,000 x g for 10 min at 4 °C.
4. Remove the supernatant and gently resuspend the pellet (thylakoids) in 50 ml of Wash Buffer (see Table 1).
5. Centrifuge the solution at 10,000 x g for 10 min at 4 °C and remove the supernatant completely. At this point, carry out the following steps in the dark, to avoid pigment oxidation.
6. Add ~20 ml of 80% acetone buffered with Na₂CO₃ (see Table 1) to extract the pigments. Leave the solution on ice for 10 min, vortexing occasionally.
7. Pellet the cellular components by centrifugation at 12,000 x g for 15 min at 4 °C.
   NOTE: If the pigments are not totally extracted, the pellet will have a green color and step 1.6 should be repeated.
8. Collect the supernatant into a separatory funnel. Add 0.4 volumes of diethylether, shake vigorously and open the valve to vent the gas.
9. Add 0.8 volumes of 0.33 M NaCl and mix vigorously. Allow ~10 min for the layers to separate. The ether phase on top contains the extracted pigments. Remove the clear lower phase.
   NOTE: If the separation is not clear, freeze and thaw the solution to improve the phase separation.
10. Remove the ether by pouring it from top of the separatory funnel into a suitable glass container. Dry by adding a spoonful of granular anhydrous sodium sulfate. Swirl the solution and allow ~5 min for the desiccant to absorb water from the ether.
    NOTE: Repeat this step if the sodium sulfate appears completely clumped together; there should be some free-floating crystals when the ether is sufficiently dried. If a water layer forms, remove this with a Pasteur pipet before adding additional anhydrous sodium sulfate.
11. Decant the ether to a new glass container, leaving the sodium sulfate solid behind.
12. Evaporate the ether in a rotary speedvac or under a stream of N₂.
13. Dissolve the pigments completely in 10 ml of 100% acetone.
14. Dilute a small amount (~3 µl) into 1 ml of 80% acetone and measure the absorption spectra and determine the Chl a/b ratio and the Chl concentration with the method described by Porra et al. (1989)¹⁵.
15. Aliquot and dry the pigments in a rotary speedvac or under N₂ stream until the acetone is completely evaporated. Store the dried pigments at -80 °C.

2. Extraction of Carotenoids from Spinach

1. Follow steps 1.1 to 1.5. At this point, carry out the following steps in the dark, to avoid pigment oxidation.
2. Resuspend the thylakoid pellet in ~50 ml 96% ethanol buffered with Na₂CO₃ (see Table 1) to extract the pigments. Leave the solution on ice for 5 min.
3. Pellet the cellular components by centrifugation at 12,000 x g for 15 min at 4 °C.
   NOTE: If the pigments are not totally extracted, the pellet will have a green color and step 2.2 should be repeated.
4. Collect the supernatant and add 0.1 volume of 80% KOH (w/v) to initiate saponification.
5. Leave the solution at 4 °C O/N, tightly capped and protected from the light.
6. Collect the solution into a separatory funnel. Add 1 volume of diethyl ether and mix gently.
7. Add 0.8 volumes of 0.33 M NaCl and mix gently. Allow ~10 min for the layers to separate. The orange ether phase on top contains the saponified carotenoids. Remove the green lower phase by draining through the stopcock of the funnel.
8. Add 3 volumes of water and mix gently to remove the potassium hydroxide. Allow the layers to separate. NOTE: If the upper phase appears cloudy, add a small amount of NaCl (e.g., 3 g of NaCl in 200 ml of solution) and swirl gently to dissolve.
9. Remove the lower phase by draining through the stopcock of the funnel.
10. Follow steps 1.10 to 1.13.
11. Dilute a small amount (~3 µl) into 1 ml of 80% acetone and measure the absorption spectra at 440 nm in 80% acetone. To determine the concentration, use the average coefficient extinction for the carotenoids (ε₄₄₀ = 255)¹⁶ in the following formula: Car [mg/ml] = (Abs_{440 nm}/225) x 11 (optical path) = 1 cm.
12. Aliquot and dry the carotenoids in a speedvac or under N₂ stream until all diethylether has been evaporated. Store the dried pigments at -80 °C.

3. Total Pigment and Carotenoid Extraction from Chlamydomonas reinhardtii

1. Grow C. reinhardtii on solid TAP medium¹⁷ in a petri dish by spreading a small amount of liquid culture onto the surface. Grow under continuous illumination flux of 20 μmol photos PSA m^-2 sec^-1 until a green layer of cells is visible.
2. Using a sterile inoculating loop, harvest a small amount of C. reinhardtii from the solid TAP medium and put the cells into 500 ml of TAP medium¹⁷ in a 1 L flask. Grow the culture at 25 °C with 170 rpm agitation under a continuous illumination flux of 20 μmol photos PSA m^-2 sec^-1.
3. After 5-6 days, the culture should reach the end of the logarithmic phase (6 x 10⁶ cell/ml or 2-2.5 optical density at 750 nm). Centrifuge the culture at 4,000 x g for 15 min at 4 °C.
4. For total pigment extraction, follow steps 1.6 to 1.15.
5. The yield of total pigment extract starting from 500 ml of full growth culture of C. reinhardtii is around 5 ml of solution with a concentration of 0.5 mg chl a+b / ml.
6. For carotenoids extraction, follow steps 2.2 to 2.12.

4. Purification of Inclusion Bodies

1. Clone the coding sequence of the LHC protein of interest into an expression vector that results in a fused C-terminal His tag using standard molecular biology procedures. Transform this construct into E. coli host strain such as BL21 (DE3).
2. Prepare Lysis buffer, Detergent buffer, Triton buffer, TE (Table 1), 1 M Isopropyl β-D-1-thiogalactopyranoside (IPTG) and LB medium¹⁸ with the appropriate antibiotics.
3. Pick a single E. coli colony containing the expression clone from a freshly streaked plate into ~5 ml of LB medium with the appropriate antibiotics using standard procedures⁶. Grow at 37 °C with 220 rpm agitation for at least 16 hr.
4. Add 2.5 ml of the O/N culture into a 1 L Erlenmeyer flask with 250 ml of LB supplemented with the appropriate antibiotic.
5. Grow the cells for 2-3 hr (or until the OD₆₀₀ is ~0.6) at 37 °C at 220 rpm.
6. Add IPTG to a final concentration of 1 mM. Continue to grow the cells at 37 °C with 220 rpm 3-4 hr.
7. Centrifuge the culture for 10 min at 5,000 x g at 4 °C in a pre-weighed centrifuge tube. Discard the supernatant thoroughly and determine the weight the pellet by weighing again and subtracting the centrifuge tube weight.
8. Resuspend the E. coli cell pellet in 0.8 ml/g of Lysis buffer by vigorous vortexing.
   NOTE: Alternatively, the cell pellet can be frozen at -80C for later use. If starting with a frozen pellet, allow to thaw completely before adding the lysis buffer.
9. Add 2 mg of lysozyme per gram of cells, and incubate on wet ice with occasional vortexing for 30 min.
10. Add 20 μg/ml DNAse, 10 mM MgCl₂, 1 mM NaCl, 20 µg/ml RNAse. Vortex and put on ice for 30 min.
11. Add 2 ml of cold Detergent buffer per gram of cells. Mix well and keep RT for 5 min.
12. Transfer to 2 ml centrifuge tubes (split into two tubes if necessary). Centrifuge for 10 min at 12,000 x g at 4 °C to pellet the inclusion bodies.
13. Add 1 ml of cold Triton buffer and completely resuspend the pellet by sonification (3 pulses x 5 sec x 50% power with 20 sec intervals). NOTE: Have the tube in a small beaker surrounded by ice water to keep it cold during the sonification. In the case of multiple tubes, combine the resuspended inclusion bodies into one tube after resuspension.
14. Centrifuge for 10 min at 12,000 x g at 4 °C to pellet the inclusion bodies.
15. Repeat step 4.13 and 4.14 two times.
16. Resuspend the inclusion bodies in 1 ml of cold TE with sonification for a final wash to remove the Triton buffer. Centrifuge for 10 min at 12,000 x g at 4 °C to pellet the inclusion bodies.
17. Resuspend the pellet in 1 ml of cold TE by sonification.
18. Assess the protein concentration by standard methods such as the Bradford assay¹⁹. Store aliquots of the inclusion bodies at -20 °C.

5. Reconstitution

This protocol typically yields 1-2 ml of reconstituted protein with an OD of 4 when absorbance is measured in the Qy region (600-750 nm). Quantity can be adjusted as desired, although care should be taken to maintain the proper ratios during the procedure.

1. Prepare the following solutions as described in Table 1: 2x Reconstitution Buffer, 20% OG, 2 M KCl, TE. Perform the following steps in dim light.
2. Resuspend 800 μg of LHC Inclusion Bodies in a total of 400 μl TE in a 2 ml microfuge tube. Add 400 μl of the 2x Reconstitution Buffer and vortex briefly.
3. Add 0.6 μl of β-Mercaptoethanol (stock 14.8 M) to have a final concentration of 10 mM. Heat the protein for 1 min at 98 °C. Vortex briefly and place at RT for 3 min.
4. Resuspend 500 μg of total dried chlorophyll pigments plus 80 μg carotenoid pigments in 30 μl 100% EtOH by vigorously vortexing for 1 min or place in a bath sonicator for 1-2 min.
5. Spin the pigment mix ~30 sec at 15,800 x g at 4 °C and confirm that there is no pellet. If there is a pellet, repeat vortexing and/or sonification. IMPORTANT: After the resuspension and spin, immediately add pigment to the protein, or it can aggregate and will need to be resuspended again.
6. Slowly add the pigment mix to the cooled protein while vortexing. Continue to vortex 5-10 sec and place tube on wet ice. Be careful not to vortex too vigorously as the protein can overflow the top of the tube.
7. Add 94 μl of 20% Octyl β-D-glucopyranoside (OG) (final concentration 2%), vortex briefly and keep on ice 10 min.
8. Add 90 μl of KCl 2 M (final concentration 150-200 mM), vortex briefly and keep on ice 20 min. NOTE: column preparation (Section 6) can be initiated at this time.
9. Spin for 10 min at 15,800 x g at 4 °C. Remove the supernatant without disturbing the pellet (precipitated LDS) to a 10 ml tube. Keep cold and protected from light.

6. Nickel Column Purification

1. Prepare the following solutions as described in Table 1: OG buffer, OG rinse buffer, Elution buffer.
2. Connect a Ni-Sepharose column (1 ml) or equivalent to a peristaltic pump ensuring that no air gets inside the column during this step and the following steps.
3. Set the speed of the pump to 1 ml/min and rinse the column with 5-10 ml of water to remove the storage solution.
4. Equilibrate the column with 3-4 ml of OG buffer.
5. Add 3-4 ml of OG buffer to the protein sample and load to the column. NOTE: if the protein has been sitting on ice for longer than 10 min after removal of LDS, spin again at 15,800 x g at 4 °C for 1 min to remove any additional LDS precipitation.
6. Rinse the column with 5 ml of OG buffer.
7. Rinse the column with 2 ml of OG rinse buffer.
8. Elute the bound protein with 3 ml elution buffer. Collect the green elute which contains the reconstituted protein. NOTE: This is usually about 1 ml in total.

7. Sucrose Gradient Centrifugation

1. Prepare the following solutions as described in Table 1: Sucrose solution, 0.06% β-DM, 0.01 M HEPES, pH 7.6.
2. Fill ultracentrifuge tubes with the sucrose solution and freeze at -20 °C O/N or -80 °C for at least 1 hr.
3. Remove the tube from freezer and allow to thaw undisturbed at 4 °C. NOTE: The freeze/thaw process creates a gradient from 0.1 to 1 M sucrose. A 15 ml tube typically thaws in about 3 hr.
4. Carefully remove from the top the same volume as the green fraction eluted from the nickel Sepharose column in step 6.8. Then load the reconstituted sample on top slowly to avoid disturbing the gradient.
5. Balance tubes and spin at 200,000 x g at 4 °C in an ultracentrifuge using a SW-41 or SW-60 swinging bucket rotor for 18 hr, set to slow acceleration and stopping without brakes.
6. Carefully take out the gradient from the tube holder with forceps. Use a syringe with a long needle that has a blunt opening to collect the fraction from the top. NOTE: Alternately, collect fractions from the bottom by piercing the tube with a needle and collecting drops.