| Folding of LHC-II monomers:
• heat 0.7 mg inclusion bodies in 1.4 ml Reaction Buffer ( 100 mM Tris-Cl, pH 9.0, 2% (w/v) LDS, 12.5 % (w/v) sucrose) for 5 min at 95ºC
• cool to room temperature (~ 15 min)
• add 0.15 ml beta-mercaptoethanol (100 mM)
• cool on ice.
• inject photosynthetic Pigments (0.5 mg Chla/Chlb~3, and 0.15 mg Carotenoids in 0.15 ml EtOH) mix immediately
• keep on ice 20 min
• add 0.15 ml 10 % (w/v) beta-octyl-glucoside and mix
• add 0.2 ml of 3M KCl to precipitate the Dodecylsulfate
• keep 10 min on ice.
• spin down 5 min maxspeed tabletop
• the supernatant is now light green and contains the LHC-II monomers
you can easily scale up to a final volume of 150 ml. Folded monomers can be stored at –20° C. for later use
Trimerformation on a Ni chelating column
• prepare small columns with 1ml chelating Sepharose (Pharmacia)
• wash with some water
• load 1 ml o.3M NiCl2
• wash with 3 ml ml 50mM Tris pH 7.5
• load folded LHC-II monomers
• wash with 1 ml OG buffer (1% (w/v) beta-octyl-glucoside, 0.1 M Tris pH 9.0, 12.5% (w/v) sucrose)
• wash with 1 ml TX buffer (0.05% (v/v) Triton X-100, 0.1 mg/ml L-phosphatidyl-D,L-glycerol dipalmitoyl (PG), 0.1 M Tris pH 7.5)
• wash with Elution buffer (0.1% (w/v) beta-dodecyl-maltoside, 0.3 M Imidazol pH 7.5) until green fraction starts eluting (about 0.6ml)
• elute green fraction with another 1.2 ml Elution buffer
the eluted green fraction contains a mixture of refolded LHC-II monomers and trimers with some free pigments
Sucrose gradient centrifugation to separate monomers, trimers and free pigments
• freeze sucrose solution (20.5% (w/v) sucrose, 50 mM Tris 7.5, 0.05% (w/v) beta-dodecyl-maltoside) in SW40 Ti rotor tubes
• thaw sucrose solution slowly at 4° C.
• load folded LHC-II
• centrifuge overnight in SW40 Ti rotor (20h, 40000 rpm, 200000 g, 4° C.)
• use a syringe to harvest trimers (lowest of the three bands)
• store protein at –20° C.
Good luck and have fun
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