Journal of Biotechnology 101 (2003) 289 /293 www.elsevier.

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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, Italy b Laboratorio Provinciale di Biologia Marina, Molo Pizzoli (Porto), Bari, Italy c 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 batchwise 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; Purication; Hydroxyapatite

1. Introduction Phycoerythrin is a major light-harvesting pigment of red algae. R-Phycoerythrin (R-PE) is an oligomeric protein of 240 kDa, with 6 a (about 20 kDa), 6 b (about 20 kDa), and 1 g (about 30 kDa) subunits. R-PE is commonly used as a fluorescent label in immunology and cell biology (Oi et al.,
* Corresponding author. Tel.: /39-0971-20-5563; fax: /390971-20-5687. E-mail address: riccio@unibas.it (P. Riccio).

1982; Kronik, 1986) and in flow cytometry (Hardy, 1983; Wilson et al., 1991), but is also used 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 isoelectrofocusing (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 chromatography and gel filtration (Hilditch et al., 1991; Thammapalerd et al., 1996) or by prepara-

0168-1656/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-1656(03)00002-6

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Fig. 1. Flow sheet of R-phycoerythrin purication from Corallina elongata. Protein content was determined using the Bio-Rad Bradford reagent with BSA as a standard. * S.A. (specic activity) /A566 nm/mg prot. ** P.I. (purity index) /A566 nm/A280 nm.

tive polyacrylamide gel electrophoresis (GallardIrmouli 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 elongata , a new source of R-PE. This procedure is based on adsorption chromatography on hydroxyapatite (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 resuspended 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

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Fig. 2. Absorption spectrum of puried R-phycoerythrin. Both the absorption spectrum and the purity indexes of puried Rphycoerythrin were determined using an UV-Vis spectrophotometer Ultrospec 2000 (Pharmacia Biotech).

min and centrifuged for 10 min at 3000 /g at 4 8C. The supernatant, red coloured, was passed through a 1.0 mm cellulose nitrate membrane filter (Whatman), adsorbed on a hydroxyapatite column (16 /80 mm) connected to a FPLC system (Pharmacia 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 10 mM NaPi, 100 mM NaCl, pH 7.0 at a flow rate of 1.0 ml min 1. 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/A495 B/1.5; A620/ A566 B/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 index as 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 Bphycoerythrin (B-PE) exhibits only a slight shoulder. When A566/A495 B/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 were obtained 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/

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2. Conclusions The aims of our study were to find both a local natural source of R-PE and a new, simple procedure for its purification. R-PE is commonly extracted from algae different from C. elongata . Some examples are: Ceramium isogonum (Qian et al., 1991); Corallina officinalis (Hilditch et al., 1991); Gracilaria longa (DAgnolo et al., 1994); Gracilaria fisheri (Thammapalerd et al., 1996); Palmaria palmata (GallardIrmouli 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 obtained 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 an attractive proposition for the production of RPE and its purification.

Fig. 3. SDS gel electrophoresis. Lanes: 1, standard proteins; 2, extract after ltration; 3, hydroxyapatite eluate; 4, puried Rphycoerythrin. SDS polyacrylamide gel electrophoresis was performed using a Mini-Protean II (Bio-Rad) on discontinuous gels according to Sch.a gger and von Jagow (1987), using a 4% spacer gel and 10% running gel. Proteins were solubilized with a medium containing 4% SDS, 2% b-mercaptoethanol, 12% glycerol, 0.01% bromophenol blue, 50 mM Tris /HCl at pH 6.8. The amount of protein loaded to the gel was 15 mg. Proteins were stained in 0.2% Coomassie blue R-250 and 0.05% Coomassie blue G-250 in methanol:acetic acid:water (4:1:4, v/ v/v). Densitometric analysis was carried out using an Ultroscan XL Enhanced Laser spectrodensitometer and the Gel Scan XL software (Pharmacia Biotech).

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 the worst. 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 batchwise 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.

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