Immunological properties of sedoheptulose-1,7-bisphosphatase from Chlamydomonas sp. W80

Article abstract of DOI:10.1271/bbb.69.848

Here we report on the production of functional recombinant SBPase of Chlamydomonas sp. W80 in Escherichia coli and the one-step purification of a polyhistidine-tagged fusion protein. The polyclonal antibody was raised against purified recombinant enzyme and cross-reacted with crude SBPase from Chlamydomonas, spinach, tobacco, and Arabidopsis leaves. Further, we investigated the levels of protein and activity of SBPase in different tissues of Arabidopsis plants.

Full text of DOI:10.1271/bbb.69.848

Biosci. Biotechnol. Biochem., 69 (4), 848–851, 2005
Note
Immunological Properties of Sedoheptulose-1,7-bisphosphatase
from Chlamydomonas sp. W80
Masahiro TAMOI,y Miki NAGAOKA, and Shigeru SHIGEOKA
Department of Food and Nutrition, Faculty of Agriculture, Kinki University,
3327-204 Nakamachi, Nara 631-8505, Japan
Received November 29, 2004; Accepted December 21, 2004
Here we report on the production of functional
recombinant SBPase of Chlamydomonas sp. W80 in
Escherichia coli and the one-step purification of a
polyhistidine-tagged fusion protein. The polyclonal anti-
body was raised against purified recombinant enzyme
and cross-reacted with crude SBPase from Chlamydo-
monas, spinach, tobacco, and Arabidopsis leaves. Fur-
ther, we investigated the levels of protein and activity of
SBPase in different tissues of Arabidopsis plants.
fructose-1,6-/sedoheptulose-1,7-bisphosphatase (FBP/
SBPase) in the chloroplasts of tobacco plants results in
enhanced photosynthesis efficiency, sugar content, plant
size, and dry matter.9) These findings strongly indicate
that SBPase is positioned at the RuBP regenerative
stages and is highlighted as one of the enzymes that
contribute to the control of the carbon flux of the Calvin
cycle.
However, little information on the molecular proper-
ties of the enzyme has been reported for photosynthetic
organisms, because there has been no antibody against
SBPase from photosynthetic organisms, including high-
er plants, and recently, we have not been able to obtain
SBP as a substrate from any commercial source.
Recently we analyzed the nucleotide sequences of
cDNA encoding SBPase from halotolerant Chlamydo-
monas sp. W80 (C. W80) cells, which showed approx-
imately 65% homology to other SBPases from higher
plants.10)
Taking the present situation concerning SBPase into
consideration, we produced functional recombinant
SBPase as a polyhistidine-tagged fusion protein in
E. coli by one-step affinity chromatography and the
antibody raised against the recombinant SBPase. Then
we studied the immunological properties and levels of
protein and activity of SBPase in different tissues of
Arabidpsis plants.
For construction of a plasmid to express SBPase, a
cDNA fragment encoding the mature form was ampli-
fied with PCR using the oligonucleotide primers P-1, 50-
ACGTGCATATGGGCGACTCC-30 and P-2, 50-CACT-
CGCTGCACAGAGGGCA-30. Amplified fragment was
subcloned into the pSTBlue T-vector. Following trans-
formation of E. coli strain DH5a cells, a candidate clone
was isolated and sequenced across the region of interest
with DNA sequencing in order to establish the fidelity of
SBPase construction. The plasmid was digested with
Nde I and Bam HI, and the resulting 1.0-kbp DNA
fragment was cloned in frame behind an N-terminal 6 ꢀ
His-tag into the pET16b vector (Novagen, Madison, WI,
Key words: Chlamydomonas sp. W80; sedoheptulose-
1,7-bisphosphatase; antibody; Calvin cycle;
Arabidopsis
The photosynthetic carbon reduction cycle (the Calvin
cycle) is the primary pathway of carbon fixation in
photosynthetic organisms, including higher plants. The
photosynthetic carbon metabolism in higher plants is
thought to be one of the determining factors in plant
growth and crop yield. Sedoheptulose-1,7-bisphos-
phatase (SBPase, EC 3.1.3.37) catalyzes the irreversible
dephosphorylation of sedoheptulose 1,7-bisphosphate
(Sed 1,7-P2) to sedoheptulose 7-phosphate (Sed 7-P),
and is responsible for the regeneration of CO2-acceptor
ribulose-1,5-bisphosphate (RuBP) in the Calvin cycle.
SBPase, as well as other thiol-modulated enzymes in the
Calvin cycle, are regulated by a ferredoxin/thioredoxin
system in the stroma of chloroplasts in photosynthetic
organisms.1)
In order to elucidate the limiting step of the Calvin
cycle and its effect on carbon allocation and crop yield
(productivity), many researchers have generated trans-
genic plants with altered enzyme activities involved in
the Calvin cycle with antisense technology.2,3) As for the
result, transgenic plants with a smaller decrease in the
activity of FBPase or SBPase showed a significant
reduction in photosynthetic capacity. This reduction in
photosynthesis might result from a reduction in the
regenerative capacity of the Calvin cycle.4–8) Recently,
we reported that overexpression of cyanobacterial
y
To whom correspondence should be addressed. Tel: +81-742-43-8196; E-mail: tamoi@nara.kindai.ac.jp
The nucleotide sequence data for sedoheptulose-1,7-bisphosphatase reported in this paper will appear in the GSDB, DDBJ, EMBL, and NCBI
nucleotide sequence databases under accession no. AB035313.

Immunological Properties of Chlamydomonas SBPase
849
U.S.A.) digested with the same restriction enzymes.
The construct obtained, designated pETCW80sbp, was
introduced into E. coli BL21(DE3)pLysS. The E. coli
cells transformed with pETCW80sbp were grown in
5 ml-LB medium supplemented with 50 mg/ml ampicil-
lin and 34 mg/ml chloramphenicol at 37ꢁC overnight.
The culture was then inoculated with the final 1.0 liter-
LB culture. When the culture reached an absorbance of
0.6 at 600 nm, 0.4 mM IPTG was added and the bacteria
were grown for a further 6 h at 37ꢁC. Cells were
harvested by centrifugation at 6;000g for 10 min and the
pellets were kept frozen at ꢂ20ꢁC.
nearly 30% of the total protein in the E. coli cells. The
enzyme activity of recombinant SBPase in the soluble
fractions was 7:15 ꢃ 0:61 mmol/min/mg protein. En-
zyme activity was not found in the insoluble fractions.
All procedures for purification of the recombinant
enzyme were carried out at 4ꢁC. E. coli cells (0.8 g wet
wt) were suspended in 10 ml of 20 mM Tris–HCl buffer
(pH 7.4) containing 500 mM NaCl, sonicated (10 kHz)
for a total of 1 min with 5 intervals of 10 sec each, and
centrifuged at 25;000 ꢀ g for 20 min. The resulting
supernatant was loaded onto a HisTrap chelating HP
column (1 ml) according to the manufacturer’s instruc-
tions (Amersham Bioscience, Piscataway, NJ, U.S.A.).
The purification procedure yielded approximately
2.8 mg recombinant protein of C. W80 SBPase.
SDS–PAGE of the purified enzyme showed only one
detectable protein band (Fig. 1, lane 4).
The purified recombinant C. W80 SBPase (44 mg) was
suspended in 2.2 ml of 100 mM Tris–HCl (pH 8.0) and
emulsified with the same volume of Freund’s complete
adjuvant. Eleven BALB/c male mice (5 weeks old) were
immunized twice with subcutaneous injections (first, 3-
mg injection per mouse; second, 1-mg injection per
mouse) with a 4 week interval. Ten d after the second
injection, blood was collected from the tail vein of each
mouse and the serum was separated from the blood. The
sera were tested with an enzyme-linked immunosorbent
assay, and blood was collected from the orbital vein of a
high-titer mouse.
Overexpression of the recombinant SBPase was
confirmed by assay of the recombinant enzyme activity
and by SDS–PAGE analysis of the enzyme protein in
the soluble fractions prepared from the crude extracts of
E. coli cells. The activity of SBPase was determined
with a discontinuous assay in which the Pi release was
measured at fixed intervals. The reaction mixture (final
volume 2.1 ml) contained 100 mM Tris–HCl (pH 8.0),
1.5 mM Sed 1,7-P2, 10 mM MgCl2, and the enzyme.11)
SBP was synthesized from dihydroxyacetone phos-
phate (DHAP) and erythrose 4-phosphate (Ery 4-P) with
an enzyme reaction with recombinant cyanobacterial
aldolase (CI-FBA).12) The reaction mixture (final vol-
ume 10 ml), containing 100 mM Tris–HCl (pH 7.5),
300 mmol of DHAP, 300 mmol of Ery 4-P, and
15 mmol/min of CI-FBA, was incubated for 24 h at
27ꢁC. The mixtures were subjected to a Dowex 1
column (2:6 ꢀ 10 cm) and then developed with a
240 ml-linear gradient of (NH4)2CO3 (0–1 M) at flow
rate of 0.2 ml/min. The fractions containing Sed 1,7-P2
were confirmed by measuring SBPase activity.
The polyclonal antibody was cross-reacted with the
recombinant SBPase purified from E. coli and the native
enzyme from C. W80 cells (Fig. 2). Interestingly, the
polyclonal antibody showed an ability to cross-react
with crude homogenates from spinach, tobacco, and
Arabidopsis leaves and to react specifically with
approximately 40-kDa proteins corresponding to
SBPase in these plants. In contrast, the antibody failed
to cross-react with the homogenate of cyanobacteria,
SDS–PAGE and immunoblotting were carried out
according to the methods described previously.13) As
shown in Fig. 1, we observed a protein band corre-
sponding to recombinant SBPase with a molecular mass
of 41.6 kDa calculated from the deduced amino acid
sequence of cloned SBPase and the His-tag sequence in
the pET16b vector. Recombinant SBPase accounted for
Fig. 2. SDS–PAGE (A) and Immunoblot Analysis (B) of Various
Sources Using Antisera Raised against C. W80 SBPase.
Each protein (30 mg) was subjected to SDS–PAGE. Lane 1, crude
extract of C. W80; lane 2, crude extract of Synechococcus
PCC7942; lane 3, crude extract of Synechocystis PCC6803; lane 4,
crude extract of Nicotiana tabacum cv. Xanthi (leaf); lane 5, crude
extract of Spinacia oleracea (leaf); lane 6, crude extract of
Arabidopsis thaliana (rosetta leaf); lane 7, purified recombinant
CW80SBPase.
Fig. 1. Expression of C. W80 SBPase in E. coli.
Protein samples (corresponding to 50 mg of total protein) were
prepared from cell lysates and then analyzed with 12.5% SDS–
PAGE. Lane 1, molecular mass standards (Pharmacia); lane 2, pET-
16b-transformed E. coli; lane 3, pETCW80sbp-transformed E. coli
after induction with 0.4 mM IPTG; lane 4, purified recombinant
CW80SBPase.

850
M. TAMOI et al.
confirming that these organisms are absent from the
gene encoding an SBPase.9,10)
observed in the Calvin cycle of chloroplasts of Arabi-
dopsis plants. The present data confirm that SBPase is a
key enzyme in the primary pathway of carbon fixation in
higher plants. A detailed investigation of the substantial
contribution of SBPase to the regulation of carbon flow
in the Calvin cycle is now under way in our laboratory
using the polyclonal antibody and transgenic plants
expressing SBPase from Chlamydomonas.
It has been reported that the deduced amino acid
sequence of C. W80 SBPase showed a high homology
(65%) to other SBPases from higher plants.10) These
findings indicate that the primary structures of SBPase
between C. W80 and higher plants are similar due to the
existence of highly conserved domains, and suggest the
possibility that the polyclonal antibody raised against C.
W80 SBPase can be utilized for immunodetectional and
immunoquantification analysis of SBPase in higher
plants.
In order to investigate the expression profile of
SBPase in Arabidopsis plants, extractable proteins
prepared from the roots, stems, leaves, flowers, and
green siliques of 3- to 6-week-old plants were subjected
to immunoblot analysis using CW80SBPase polyclonal
antibody (Fig. 3). Bands corresponding to Arabidopsis
SBPase were detected in all tissues except for the roots.
The activity of the enzyme in the leaves of Arabidopsis
was 223:5 ꢃ 48:8 nmol/min/mg protein, while no ac-
tivity of the enzyme in the roots was detected. The levels
of protein and activity of SBPase were in agreement
with the transcript level in different tissues of Arabi-
dopsis.14)
Most of the enzymes involved in the Calvin cycle are
present at levels well in excess of those required to
sustain a continued rate of CO2 fixation in higher plants.
However, it is likely that levels of fructose-1,6-bis-
phosphatase (FBPase) and sedoheptulose-1,7-bisphos-
phatase (SBPase) are extremely low compared with
those of other enzymes in the Calvin cycle.15)
The activities of NADPþ-glyceraldehyde-3-phosophate
dehydrogenase, phosphoribulokinase, and FBPase in
leaves of Arabidopsis were 19:2 ꢃ 3:1, 33:2 ꢃ 6:3 and
2:68 ꢃ 0:44 mmol/min/mg chlorophyll respectively (un-
published data). Low activity of SBPase was also
Acknowledgments
This work was supported by a project grant (to S.S.)
titled ‘‘Functional analysis of genes relevant to agricul-
turally important traits in the rice genome’’ from the
Ministry of Agriculture, Forestry, and Fisheries of
Japan, and by the Japan Society for the Promotion of
Science Research for the Future Program (grant no.
JSPS-RFTF 00L01604). It was also supported in part by
‘‘Academic Frontier’’ Project for Private Universities:
matching fund subsidy from MEXT 2004–2008.
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