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Short communication
Extracting and purifying R-phycoerythrin from Mediterranean
red algae Corallina elongata Ellis & Solander
R. Rossano a, N. Ungaro b, A. DAmbrosio a, G.M. Liuzzi c, P. Riccio a,*
a Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali, University of Basilicata, Campus Macchia Romana, I-85000 Potenza,
Italyb Laboratorio Provinciale di Biologia Marina, Molo Pizzoli (Porto), Bari, Italyc Dipartimento di Biochimica e Biologia Molecolare, University of Bari, Bari, Italy
Received 8 March 2002; received in revised form 12 December 2002; accepted 19 December 2002
Abstract
R-Phycoerythrin (R-PE) is a protein acting as a photosynthetic accessory pigment in red algae (Rodophyta). This
protein has gained importance in many biotechnological applications in food science, immunodiagnostic, therapy,
cosmetics, protein and cell labelling, and analytical processes. In this paper we report on a new, one step procedure for
the extraction and purification of R-PE from a new source: the Mediterranean red algae Corallina elongata Ellis &Solander. This red algae contains mainly R-PE and is suitable for the production in culture. No other contaminating
phycobiliproteins could be detected in the extracts. The method we propose for the purification is based on the use of
hydroxyapatite, a chromatographic resin that can be produced in the laboratory at very low cost and can be used batch-
wise with large amounts of extracts, alternative to chromatography, and therefore can be scaled up. Both the yield and
the purity of R-PE are very good.
# 2003 Elsevier Science B.V. All rights reserved.
Keywords: Algae; Corallina elongata ; Phycoerythrin; Purification; Hydroxyapatite
1. Introduction
Phycoerythrin is a major light-harvesting pig-
ment of red algae. R-Phycoerythrin (R-PE) is an
oligomeric protein of 240 kDa, with 6 a (about 20
kDa), 6b (about 20 kDa), and 1g (about 30 kDa)
subunits. R-PE is commonly used as a fluorescent
label in immunology and cell biology (Oi et al.,
1982; Kronik, 1986) and in flow cytometry
(Hardy, 1983; Wilson et al., 1991), but is alsoused as a natural food dye (Mille-Claire et al.,
1993; Qian et al., 1991; DAgnolo et al., 1994) and
as a marker in gel electrophoresis and isoelectro-
focusing (Araoz et al., 1998). It has also been
shown that it could be used as a measure of peroxy
radical damage (DeLange and Glazer, 1989).
R-PE can be purified either by precipitation
with ammonium sulphate, by ion-exchange chro-
matography and gel filtration (Hilditch et al.,
1991; Thammapalerd et al., 1996) or by prepara-
* Corresponding author. Tel.: /39-0971-20-5563; fax: /39-
0971-20-5687.
E-mail address: [email protected](P. Riccio).
Journal of Biotechnology 101 (2003) 289/293
www.elsevier.com/locate/jbiotec
0168-1656/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved.
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tive polyacrylamide gel electrophoresis (Gallard-Irmouli et al., 2000). The use of hydroxyapatite in
association with gel filtration and precipitation
with ammonium sulfate has also been reported
(MacColl et al., 1996).
In this paper we have set up an alternative
procedure for the purification of R-PE extracted
from the Mediterranean red algae Corallina elon-
gata , a new source of R-PE. This procedure is
based on adsorption chromatography on hydro-
xyapatite (HA), a form of calcium phosphate
Ca10(PO4)6(OH)2 (Tiselius et al., 1956; Riccio,1989), and gel filtration. C. elongata was collected
in the southern Adriatic Sea near the town of Bari,
Italy. Algae samples were washed with deionized
water, lyophilised in small aliquots and stored at
/70 8C until use.
The purification procedure is shown in Fig. 1.
Twenty-five grams of lyophilised algae were re-
suspended in three volumes of a buffer containing
10 mM sodium phosphate (NaPi)/100 mM NaCl/
pH 7.0, blended in a cold Waring Blendor for 3
Fig. 1. Flow sheet of R-phycoerythrin purification from Corallina elongata. Protein content was determined using the Bio-Rad
Bradford reagent with BSA as a standard. * S.A. (specific activity)/A566 nm/mg prot. ** P.I. (purity index)/A566 nm/A280 nm.
R. Rossano et al. / Journal of Biotechnology 101 (2003) 289 /293290
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min and centrifuged for 10 min at 3000/g at
4 8C. The supernatant, red coloured, was passedthrough a 1.0mm cellulose nitrate membrane filter
(Whatman), adsorbed on a hydroxyapatite column
(16/80 mm) connected to a FPLC system (Phar-
macia Biotech) and eluted with 50 mM NaPi/0.1
M NaCl/pH 7.0. After adsorption the upper
portion of the HA column became red and the
non-adsorbed, pass-through, fraction appeared
yellow. The red coloured fractions were collected,
concentrated by ultrafiltration on YM10 Amicon
membranes, and applied to a Superdex 75 gel
filtration FPLC column. The eluting buffer was 10mM NaPi, 100 mM NaCl, pH 7.0 at a flow rate of
1.0 ml min1. The eluate showed two peaks.
Fractions in the first peak, with purity index
A566/A280 greater than 5.0, were collected. The
absorption spectrum of purified R-PE showed the
characteristic peaks at 495 and 566 nm (Fig. 2).
Purity of R-PE was estimated by means of three
indexes: A566/A280/5.3; A566/A495B/1.5; A620/
A566B/0.005. The 1st, A566/A280, is indicative of
the purity of the preparation with respect to many
contaminating proteins. This index should be
higher than 5.3. Our R-PE showed a purity indexas high as 6.67, corresponding to a 61-fold
enrichment of the specific activity (A566/mg prot.).
The 2nd, A566/A495, indicative of the identity of
the purified pigment, was 1.44. R-PE has a strong
secondary absorbance peak at 495 nm, where B-
phycoerythrin (B-PE) exhibits only a slight
shoulder. When A566/A495B/1.5, the pigment is
not significantly contaminated with B-PE. Finally,
the index A620/A566 of our R-PE preparation was
0.001.
About 15 mg of pure phycoerythrin wereobtained from 25 g of lyophilised algae. Analysis
of purified protein by SDS gel electrophoresis (Fig.
3) gave three bands. The upper band should
correspond to a 55 kDa heterodimer of the a/b
and g subunits, while the second and the third
bands correspond to the g (30 kDa) and a/b
subunits (20 kDa).
The stability of R-PE (1 mg prot. ml1; 10 mM
NaPi; 100 mM NaCl; pH 7.0) was monitored over
time in different conditions (/20 8C/4/8 8C/
Fig. 2. Absorption spectrum of purified R-phycoerythrin. Both the absorption spectrum and the purity indexes of purified R-
phycoerythrin were determined using an UV-Vis spectrophotometer Ultrospec 2000 (Pharmacia Biotech).
R. Rossano et al. / Journal of Biotechnology 101 (2003) 289 /293 291
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room temperature; in the dark and in the light, and
at pHs between 3.0 and 10.0) measuring the
absorption spectra and the absorbance at 566 nm
after 1:10 dilution. A566 clearly decreased with the
breakdown of the chromophore or with the
aggregation and precipitation of R-PE. After 35
days the sample stored in the dark at 4 8C showed
the greatest stability, and that at /20 8C theworst. Freezing and thawing clearly induced the
formation of red aggregates. When exposed to the
light, R-PE showed a higher trend in losing its
chromophore, and the effect of light was even
higher after 15 days. R-PE was very stable at pHs
in the range 6.0/8.5, but at low pHs showed a
marked decrease of A566. Alternatively, a batch-
wise procedure was used in the first step by adding
HA to the deep red supernatant at the ratio of 0.1
g HA ml1 extract.
2. Conclusions
The aims of our study were to find both a local
natural source of R-PE and a new, simple proce-dure for its purification.
R-PE is commonly extracted from algae differ-
ent from C. elongata . Some examples are: Cer-
amium isogonum (Qian et al., 1991); Corallina
officinalis (Hilditch et al., 1991); Gracilaria longa
(DAgnolo et al., 1994); Gracilaria fisheri (Tham-
mapalerd et al., 1996);Palmaria palmata (Gallard-
Irmouli et al., 2000).
Our results indicate that C. elongata could be a
good alternative source of R-PE. In addition to its
good phycoerythrin content, the choice of C.elongata appears to be very convenient because
no other contaminating phycobiliproteins could be
detected in the extracts. This makes the R-PE
purification procedure presented in this paper very
simple and easily reproducible. When compared
with other purification procedures, our method
which is useful for scale up gives a product with a
high enrichment of specific activity (A566/mg
prot.), one of the highest purity indexes, a good
yield and satisfying stability at 4 8C in the dark
and at pH 6.0/
8.5.The purity index obtained in this study is much
better than those reported earlier for Phyllophora
antarctica (MacColl et al., 1996) or for Palmaria
palmate (Gallard-Irmouli et al., 2000) who ob-
tained purity indexes of 4.4 and 3.2, respectively.
Furthermore, our procedure is less laborious than
those reported by other authors (Hilditch et al.,
1991; MacColl et al., 1996; Thammapalerd et al.,
1996), requiring two or three purification steps.
The possibility of growing C. elongata under
laboratory conditions (Vergara and Niell, 1993)
and applying our one step procedure is anattractive proposition for the production of R-
PE and its purification.
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