Development of Cell-Free Systems - Chlorophyll Biosynthesis and Technological Applications - page 159

EDTA, and various amounts of glycerol and CHAPS as indicated in specific
experiments. The pH was adjusted to 7.7 at room temperature. Solubilization was
carried out on ice with continuous stirring for 30 min. After 30 min, the suspension
was centrifuged at 100,000 g for 1 h in a Beckman Ti80 fixed angle rotor at 2 C.
The resulting supernatant containing 1-2 mg solubilized membrane protein per ml
was stored at

80 C until use. No detectable loss of 4VCR activity was observed
after several months of storage at

80 C (Kolossov and Rebeiz 2001 ).

Chromatography of Solubilized Etioplast Membrane Proteins
on DEAE-Sephacel

Partial purification of 4VCR was achieved by chromatography on DEAE-Sephacel.
About 7 ml of the solubilized membrane protein fraction were applied to a 5 ml
column of DEAE-Sephacel which was pre-equilibrated with a solution of 5 %
glycerol, 20 mM Tris-HCl, 0.1 mM DTA, and 4 mM CHAPS, adjusted to a room
temperature pH of 7.7. Elution of 4VCR activity was achieved at a speed of 0.4 ml
per min, with a linear 0.02 M ammonium sulfate gradient dissolved in the
pre-equilibration buffer. Two-ml fractions were collected, and 4VCR activity was
monitored. The active fractions were pooled and concentrated in disposable Millipore
Centriplus-e0 Centrifuge cartridges, until a retentate volume of 1.5-2.0 ml was
collected. The retentate contained about 1-1.5 mg solubilized protein per ml
(Kolossov and Rebeiz 2001 ).

Further Purification of 4VCR Activity on Cibacron Blue 3GA-1000 Agarose

Further purification of 4VCR activity was achieved by chromatography on Cibacron
Blue 3GA-1000 agarose. This resin acts as a non-specific affinity medium. The
column (1.5 ml bed volume) was made up of a 5 ml disposable pipette tip. It was
pre-equilibrated with column buffer that consisted of 20 mM Tris-HCl, 0.1 mM
EDTA, 5 % glycerol, 200 mM ammonium sulfate and 2.5 mMCHAPS, adjusted to a
room temperature pH of 7.7. After applying the concentrated retentate (see above) to
the Cibacron column, the column was washed with 3 bed volumes of column buffer.
4VCR activity was eluted with column butter adjusted to 5 mM CHAPS concentra-
tion (Kolossov and Rebeiz 2001 ).

Purification of Solubilized 4VCR

Solubilization of 4VCR resulted in 1.5-2.0-fold purification (Kolossov and Rebeiz
2001 ). Further purification was achieved by column chromatography on DEAE-
Sephacel. DEAE is a weak base that acquires a net positive charge when ionized.
It

therefore binds and exchanges anions. An additional two to threefolds