Purification and Characterization of Cadmium-Binding Protein fromUnicelluar Alga Chlorella sorokinian

Naoto Yoshida, Kazushige Ishii, Tomoko Okuno, Kazunori Tanaka

Department of Biochemistry and Applied Biosciences, University of Miyazaki, 1-1 Gakuen Kibanadai-Nishi, Miyazaki, 889-2192, Japan

Received: 26 September 2005 / Accepted: 24 January 2006

Abstract. The unicellular green alga Chlorella sorokiniana ANA9 is highly resistant to heavy metals,and its metal-binding proteins are induced in the presence of cadmium. A novel cadmium-bindingprotein in C. sorokiniana cultured in 100 mg/l cadmium ions for 4 days was isolated and characterized.The crude protein extract was obtained by cell disruption and partly purified by ammonium sulfateprecipitation. After purification by anion-exchange chromatography with diethylaminoethyl (DEAE)-Sepharose CL-6B, the protein was further purified by gel filtration with Sephacryl S-100, followed bySephadex G-75. The molecular weight of the purified protein was determined to be 11.5 kDa by sodiumdodecyl sulfate–polyacrylamide gel electrophoresis. The cadmium binding capacity of the purifiedprotein was 119 lg/mg. The involvement of thiol coordination in metal-ion binding was confirmed bymeasuring the ultraviolet spectrum. This article is the first to describe the metallothionein-like cadmium-binding protein from Chlorella species, the expression of which is induced by cadmium exposure.

Metallothioneins are low molecular–weight, Cys-richproteins that are ubiquitous in eukaryotic organisms.Since their first description as cadmium- and zinc-binding proteins in horse kidneys, metallothionein genesand proteins have been characterized from variousorganisms [5, 12]. In the plant kingdom, several me-tallothionein-like, including those from peas [3], maize[2], Arabidopsis species [15, 16] and Sambucus nigra[1] genes, have been recently characterized. Because ofthe nature of its metal-binding activity and induction byheavy-metal ions, metallothionein is strongly believed toplay a role in metal metabolism or detoxification [5].

A unicellular alga displaying a high growth rateunder heterotrophic conditions was recently isolatedfrom soil and identified as C. sorokiniana ANA9 [14].The isolated alga was found to be highly resistant toheavy metals such as cadmium, which had a minimalinhibitory concentration of 4 mM. This alga was capableof taking up the heavy-metal ions Cd2+, Zn2+, andCu2+at 43.0, 42.0, and 46.4 lg/mg dry weight, respec-tively [14]. Growth inhibition of Oryza sative shoots

caused by cadmium in hydroponic medium was com-pletely prevented by addition of isolated Chlorella cells.However, no protein or genetic data regarding metallo-thioneins from Chlorella sources have been reported,although a large number of studies have focused on theability of microalgae to sequester metal from the envi-ronment [4, 7, 8] and on the cellular changes induced,e.g., lipid composition, by the presence of metals. Thepresent study described the isolation and characteriza-tion of the first metallothionein-like cadmium-bindingprotein from C. sorokiniana ANA9 and will thus in-crease understanding of the mechanisms used by theseorganisms to handle heavy metals.

Materials and Methods

Detection of cadmium-binding protein. C. sorokiniana ANA9 wascultured in 50 ml YPD broth (5 g yeast extract, 10 g peptone, 10 gglucose, and 1000 ml water, pH 7.0) supplemented with or without100 lg/ml cadmium at 30�C for 96 hours. Cells were collected bycentrifugation, suspended in 10 ml 20 mM Tris-HCl buffer (pH 8.8)containing 0.15 M NaCl, and homogenized by French press at 4�C.The homogenate was centrifuged at 10,000·g for 60 minutes.Supernatant was applied to a gel filtration column (Sephacryl S-100,26.7 · 70 cm) equilibrated with 20 mM Tris-HCl buffer (pH 8.8)Correspondence to: N. Yoshida; email: a04109u@cc.miyazaki-u.ac.jp

CURRENT MICROBIOLOGY Vol. 52 (2006), pp. 460–463DOI: 10.1007/s00284-005-0328-z Current

MicrobiologyAn International Journal

ª Springer Science+Business Media, Inc. 2006

containing 0.15 M NaCl, and 5-ml fractions were collected. Elutionwas performed at a flow rate of 2.5 ml/min. Cadmium concentrationand absorbance at 280 nm and 254 nm were measured in all fractions.

Protein purification. C. sorokiniana was cultured in 1000 ml YPDbroth supplemented with 100 lg/ml cadmium at 30�C for 96 hours.Cells were collected by centrifugation at 2,000·g, suspended in 50 ml20 mM Tris-HCl buffer (pH 8.8), and homogenized by French press at4�C. Homogenate was centrifuged at 10,000·g for 60 min. Ammoniumsulfate was added to the supernatant at 80% saturation. Aftercentrifugation, supernatant retaining 90% of the cadmium wasdialyzed against 20 mM Tris-HCl buffer (pH 8.8) for 24 hours.Dialysate was added to a DEAE Sepharose CL-6B column (2.4 · 40cm) previously equilibrated with the same buffer. Protein was elutedwith a linear gradient of 0 to 0.6 M NaCl, and 10-ml fractions werecollected. Elution was performed at a flow rate of 0.33 ml/min. Peaksshowing higher cadmium concentrations was pooled and concentratedusing collodion bags (Sartorius AG, Goettingen, Germany). Theconcentrate was applied to a gel filtration column (Sephacryl S-100,2.6 · 70 cm) equilibrated with 20 mM Tris-HCl buffer (pH 8.8),including 0.15 M NaCl, and 3-ml fractions were collected. Elution wasperformed at a flow rate of 0.5 ml/min. Cadmium-containing fractionswere pooled and concentrated by ultrafiltration (Ultrafree-MC,Millipore, MA) and were then added to a Sephadex G-75 column(1.5 · 60 cm) equilibrated 20 mM Tris-HCl buffer (pH 8.8) containing0.15 M NaCl, and 2-ml fractions were collected. Elution wasperformed at a flow rate of 0.1 ml/min. Cadmium concentration andabsorbance at 280 nm and 254 nm were measured in all fractions.Cadmium concentrations in eluates derived from gel filtrationchromatography and anion-exchange chromatography were measureddirectly by atomic absorption spectrometry (ShimazuAA-646,Shimadzu, Tokyo, Japan).

Internal amino-acid sequencing. Puf N-acetyl deblockingaminopeptidase (20 U) (Takara Bio, Tokyo, Japan) was added to 100pmol purified decreased protein concentrated by lyophilization to givea protein–enzyme–reaction buffer ratio of 1:1:3. The mixture wasincubated at 50�C for 90 minutes. After sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE), the protein band wasblotted onto a Sequi-Blot polyvinyl difluoride membrane (BioradHercules, CA) and applied to a Procise 494 cLC protein-sequencingsystem (Shimadzu, Tokyo, Japan) to determine the N-terminal amino-acid sequence.

Results and Discussion

To detect the cadmium-binding protein, gel filtration ofcell extracts cultured with or without cadmium wascarried out (Fig. 1). Gel filtration of cell extract fromcells cultured with cadmium yielded a large cadmiumpeak at 254 nm (fraction no. 48). In a parallel experi-ment, cell extract from cells cultured without cadmiumwas subjected to gel filtration, but no peak correspond-ing to cadmium was detected approximately fraction no.48 (Fig. 1). These results indicate that cadmium wasbound to the macromolecular substance(s), and thatC. sorokiniana was expressing a cadmium-biding pro-tein as a result of cadmium exposure.

We then purified the cadmium-induced protein toclarify its characteristics. The purification steps for thecadmium-binding protein are listed in Table 1. The final

Fig. 1. Gel filtration profile (SephacrylS-100) of cell extracts from C. sorokinianacultured in the absence (Cd–) or presence(Cd+) of 100 lg/ml cadmium. Protein andmetalloprotein was estimated by absorbanceat 280 and 254 nm, respectively, andcadmium concentration was determined asdescribed in the text.Open circle = absorption at OD254;closed circle = absorption at OD280;open triangle = cadmium concentration.

N. Yoshida et al.: Purification and Characterization of Cadmium-Binding Protein 461

yield of the entire expression and purification processwas approximately 0.16 mg pure metallothionein-likecadmium-biding protein/l C. sorokiniana culture. Frac-tions were analyzed by 15% SDS-PAGE according tothe method of Leamli [6]. The proteins were visualizedwith silver nitrate. Only one band was identified(Fig. 2). The molecular mass of the cadmium-bindingprotein was estimated to be approximately 11.5 kDabased on SDS-PAGE mobility.

To determine the nature of the bonds between theprotein and metal ions, we examined the optical featuresof the purified protein. On the ultraviolet spectrum of thepurified protein, the maximum absorption did not occurat 280 nm but rather at 250 to 260 nm (Fig. 3), indi-cating that sulfhydryl groups play a major role in cad-mium binding and that the protein possesses fewaromatic amino acids. The shoulder, at approximately250 to 260 nm at pH 8.8, was abolished on removal ofmetal ions at low pH (pH 2.0) and was interpreted asbeing characteristic of the bonds between the proteinsand metal ions, similar to those observed in metallo-thionein [9].

The amino terminal of the purified cadmium-bind-ing protein was blocked, probably because of acetylationof the first amino acid, and thus internal amino-acid

sequencing was performed after digestion with pepti-dase. S(K)-P-P(G)-E-Q sequences were identifiednear the N terminal of the cadmium-binding proteinfrom C. sorokiniana (Fig. 4). This internal amino-acidsequence was isologous with that from histidine triadprotein from Bordetella brochiseptica [11], zinc fingerprotein from Xenopus laevis [10], and nickel–cobalt–cadmium resistance protein from Alcaligenes xylosoxi-dans [13]. It is believed that the cadmium-bindingcapacity of the metallothionein-like protein is relatedprimarily to the presence of the imidazole groups har-bored by histidine and the sulfhydryl groups of cysteineresidues. Further experiments are planned to determine

Table 1. Purification table for cadmium-binding protein from C. sorokiniana ANA9

Purification step Protein (mg) Cd2+ (lg) Cd2+ (lg)/protein (mg) Purification

Ammonium sulfate 21.70 350 16.1 1-foldDEAE-Sepharose 2.820 199 70.6 4.38-foldSephacryl S-100 0.367 34.8 94.8 5.89-foldSephadex G-75 0.160 19.0 119 7.38-fold

Fig. 2. SDS-PAGE of crude and purified cadmium-binding protein.Crude protein (2 lg) and purified protein (0.2 lg) were electropho-resed on a 15% polyacrylamide slab gel. Lane 1 = molecular weightmarkers; lane 2 = purified protein; lane 3 = crude protein.

Fig. 3. Absorbance spectrum of purified cadmium-binding proteinfrom C. sorokiniana, which exhibited a shoulder at 250 to 260 nm,characteristic of Cd-S energy transfer at pH 8.8. Spectrum of theapoprotein was produced by acidification of the cadmium-bindingprotein to pH 2.0.

Fig. 4. Comparison of internal amino-acid sequences of cadmium-biding protein with homologous proteins in the GenBank database.C. sor = cadmium-binding protein of C. sorokiniana ANA9; B = bro,histidine triad protein of B. bronchiseptica RB50 (GenBank accessionno. CAE30561); X. lea = second-order repeats in zinc finger protein ofX. laevis (P18751); A. xyl = nickel–cobalt–cadmium resistance proteinncc B of A. xylosoxidans 31A (Q44585).

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whether the present cadmium-binding protein is in-volved in the metal resistance of C. sorokiniana. Com-parison of the responses of C. sorikiniana cultivated inpresence of zinc or copper could provide insightregarding this point.

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