Proteomic detection of changes in protein expression induced by cordycepin in human hepatocellular carcinoma BEL-7402 cells

The nucleoside analogue cordycepin (3'-deoxyodenosine, 3'-dA), one of the components of cordyceps militaris, has been shown to inhibit the growth of various tumor cells. However, the probable mechanism is still obscure. In this study, the inhibition of cell growth and changes in protein expression induced by cordycepin were investigated in BEL-7402 cells. Using the MTT assay and flow cytometry, we found that cordycepin inhibits cell viability and induces apoptosis in BEL 7402 cells. Additionally. the proteins were separated using two-dimensional polyacrylamide gel electrophoresis, and eight proteins were found to be significantly, affected by cordycepin compared to untreated control; among them, two were downregulated and six were upregulated. Of the eight proteins, six were identified with peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) after in-gel trypsin digestion. These proteins are involved in various aspects of cellular metabolism. It is suggested that the effect of cordycepin on the growth of tumor cells is significantly related to the metabolism-associated protein expression induced by cordycepin. Copyright 2008 Prous Science, S.A.U. or its licensors. All rights reserved.

Effects of azadirachtin on hemolymph protein expression in Ostrinia furnacalis (Lepidoptera : Crambidae)

The botanical insecticide azadirachtin affects a variety of biological processes. Our early work indicated that protein level and type are significantly influenced by azadirachtin in pupae of Osttiniafumacalis (Guenee) (Lepidoptera: Crambidae) because a correlation exists between protein content and azadiraebtin concentration. By use of proteomic techniques, we analyzed changes in hemolymph protein expression of 48-h-old pupae in O. furnacalis induced by azadirachtin treatment. After feeding by third instars on an artificial diet containing 10 ppm azadirachtin until pupation, 48-b-old pupae were collected, and hemolymph protein samples were prepared. They were separated by two-dimensional polyacrylamide gel electrophoresis, and six proteins were significantly affected by azadiracbtin treatment compared with an untreated control. Two of these proteins were identified by database searching with peptide mass fingerprinting by using matrix-assisted laser desorption/ time-of-flight mass spectrometry after in-gel trypsin digestion. They belong to the insect apolipophorin-III and phospboribosyltransferase family, respectively. These two proteins may function on lipid metabolism in insect hemolymph. Furthermore, fat body is the center of synthesis and secretion of hemolymph proteins. We suggest that the azadirachtin exerts its insecticidal effects on the fat body of O. furnacalis by interfering with protein expression related to hemolymph lipid metabolism.

Predominant expression and cellular distribution of fish Agr2 in renal collecting system

Anterior gradient 2 (Agr2) genes encode secretory proteins, and play significant roles in anterior-posterior patterning and tumor metastasis. Agr2 transcripts were shown to display quite diverse tissue distribution in different species, and little was known about the cellular localization of Agr2 proteins. In this study, we identified an Agr2 homologue from gibe[ carp (Carassius auratus gibelio), and revealed the expression patterns and cellular localization during embryogenesis and in adult tissues. The full-length cDNA of CagAgr2 is 803 nucleotides (nt) with an open reading frame of 510 nt encoding 169 amino acids. The Agr2 C-terminus matches to the class I PDZ-interacting motif, suggesting that it might be a PDZ-binding protein. During embryogenesis, CagAgr2 was found to be transcribed in the mucus-secreting hatching gland from tailbud stage and later in the pharynx region, swim bladder and pronephric duct as revealed by RT-PCR and whole mount in situ hybridization. In the adult fish, its transcription was predominantly confined to the kidney, and lower transcription levels were also found in the intestine, ovary and gills. To further localize the Agr2 protein, the anti-CagAgr2 polyclonal antibody was produced and used for immunofluorescence observation. In agreement with mRNA expression data, the Agr2 protein was localized in the pronephric duct of 3dph larvae. In adult fish, Agr2 protein expression is confined to the renal collecting system with asymmetric distribution along the apical-basolateral axis. The data provided suggestive evidence that fish Agr2 might be involved in differentiation and secretory functions of kidney epithelium. (C) 2009 Elsevier Inc. All rights reserved.

High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris

Penaeidins, members of a new family of antimicrobial peptides constitutively produced and stored in the haemocytes of penaeid shrimp, display antimicrobial activity against bacteria, and fungi. Here, a DNA sequence encoding the mature Ch-penaeidin peptide was cloned into the pPIC9K vector and transformed into Pichia pastoris. The transformed cells were screened for multi-copy plasmids using increasing concentrations of G418. Positive colonies carrying chromosomal integrations of the Chp gene were identified by phenotype and PCR. When transformed cells were induced with methanol, SDS-PAGE and Western blotting revealed the production of a similar to6100 Da recombinant CHP (rCHP) expression product. Large scale expression revealed that rCHP was produced at 108 mg/L under optimal conditions in the highest Chp-producing P. pastoris clone. The antimicrobial activities of rCHP were studied by liquid phase analysis, which revealed that rCHP exhibited activities against some Gram-negative and Gram-positive bacteria, but had a relatively low activity against some fungi. Purification of rCHP by cation exchange chromatography and subsequent automated amino acid sequencing revealed the presence of four additional amino acids (YVEF) at the N-terminus that belonged to the cleaved fusion signal peptide; these residues may account for the observed decrease in antifungal activity. Together, these observations indicate that rCHP is an effective antimicrobial peptide that can be successfully produced at high levels in the yeast, and therefore may be a potential antimicrobial candidate for practical use. (C) 2004 Elsevier Inc. All rights reserved.

Nifedipine调节白皮松(Pinus bungeana)花粉管蛋白质表达及其对细胞壁构建的影响

花粉管是种子植物受精过程中的雄性生殖单位的载体，由于其生长依赖于胞内Ca2+梯度，并具有典型的顶端极性生长的特点，因而成为近年来研究植物细胞相互识别、胞内和胞外信号传导理想的模式系统。裸子植物花粉与被子植物相比具有萌发时间长、生长缓慢等特点。Ca2+在裸子植物花粉萌发和花粉管生长中的作用机制目前尚不明确。本研究以裸子植物白皮松(Pinus bungeana)的花粉为材料，运用不同浓度钙通道抑制剂Nifedipine（Nif）处理，对其花粉萌发和花粉管生长进行了细胞学研究和蛋白质组学分析，以探讨Ca2+对白皮松花粉生长的调控机制，为进一步揭示裸子植物花粉萌发和花粉管生长机理提供参考。 本论文首先研究了钙通道抑制剂Nif和Ca2+螯合剂EGTA对白皮松花粉萌发和花粉管生长的影响。结果表明，用Nif处理花粉后，花粉萌发和花粉管生长均受到明显抑制。经Ca2+荧光探针Fluo-3AM标记，对Nif处理后Ca2+在花粉管中的分布模式进行了观察，发现Ca2+在正常生长的花粉管中呈梯度分布，并在其顶端的荧光最强。与对照相比，处理后的花粉管荧光强度明显减弱，且顶端Ca2+梯度消失。通过EGTA漂洗后的花粉粒，在正常培养基上萌发率较高，但其生长速率受到抑制。由此说明了细胞壁钙库对花粉管生长的抑制效应显著高于对花粉萌发的影响，是花粉管生长的限速因子。同时外加钙调素还可逆转EGTA对其花粉萌发和花粉管生长的抑制。上述结果表明，白皮松花粉萌发及花粉管生长需要外源Ca2+的内流，以及胞内形成的Ca2+浓度梯度。 用FM4-64探针标记结果发现，正常花粉管中的胞吞作用主要发生在顶端和亚顶端区域，胞吞的小泡也集中分布在这两个区域。经Nif处理后，既不影响花粉管的胞吞过程，同时胞吞发生的位置也与对照相似，只是胞吞的小泡分散于整个花粉管中。电子显微镜观察表明，各种细胞器在白皮松正常生长的花粉管中分布与被子植物存在较大差异，例如前者无明显地分区现象，不具胼胝质塞。白皮松花粉管顶端和亚顶端的壁旁体与质膜融合现象频繁发生。花粉管经Nif处理后，线粒体出现不同程度的解体和液泡化，内质网液泡化和核糖体脱落，液泡大量聚集在花粉管顶端，壁旁体与膜融合现象减少，以及花粉管壁明显变薄等。此外，通过微丝特异性探针鬼笔环肽标记结果表明，正常生长花粉管的微丝呈长轴向排列， Nif处理后微丝断裂，其断裂程度与处理浓度有关。由此可见，外源Ca2+对花粉管的胞吞无明显抑制或促进作用，但对胞吞小泡重回收可能有影响。当胞内Ca2+梯度消失后，则明显抑制了微丝骨架的聚合，进而使胞吐作用减缓，高尔基体分泌小泡聚集，多种细胞器液泡化，引起花粉管顶端膨大，细胞壁变薄，继而抑制花粉管的正常生长。 运用免疫荧光标记技术显示，正常生长的花粉管壁含有纤维素、胼胝质、果胶质和阿拉伯半乳聚糖蛋白（AGPs），其中纤维素和胼胝质在细胞壁上呈均匀分布，而酸性果胶质只存在花粉管两侧壁上，酯化果胶分布于花粉管顶端。经过Nif处理后，胼胝质和酸性果胶质均在花粉管顶端累积，而AGPs和纤维素的分布却无明显变化。另外，傅里叶红外光谱分析结果也同样支持上述结论。通过花粉管壁蛋白的SDS-PAGE分析表明，Nif对细胞壁蛋白的合成也有较大的影响。 在花粉管的钙通道受到抑制后，应用蛋白质组学技术分析其蛋白质的表达图谱，通过双向电泳已分离出约1000个蛋白质斑点，经软件分析发现，除其中50个蛋白斑点外，大部分蛋白质的表达量均未发生变化。上述50个发生变化的蛋白斑点酶切后，再经过ESI－MS/MS鉴定和质谱数据库的搜索，共鉴定出28个蛋白，其中12个为上调蛋白，16个下调蛋白，根据其主要功能分为与代谢、细胞扩展、翻译后修饰以及信号相关的蛋白。经过Nif处理后，花粉管中碳水化合物代谢能力下降，ATP的产生受到抑制，参与细胞壁多糖合成及小泡运输的蛋白，如valosin containing protein（VCP）、reversibly glycolsylated polypeptide(RGP)、UDP-glucose dehydrogenase (UDPGDH)和α-tubulin表达下调。另外，通过上述方法还鉴定出与丝氨酸/苏氨酸激酶保守结构域同源的受体蛋白激酶等。 综上所述，白皮松花粉管钙通道受到抑制后，通过影响花粉管蛋白的表达，抑制微丝微管骨架的组装，致使胞吐速度变慢，花粉管壁酸性果胶质、胼胝质等多糖分布的变化及总多糖含量的减少，最终抑制了花粉管的正常生长。

Identification of genes differentially expressed in wild type and Purkinje cell degeneration mice

Purkinje cell degeneration (pcd) mice are characterized by death of virtually all cerebellar Purkinje cells by postnatal day 30. In this study, we used DNA microarray analysis to investigate differences in gene expression between the brains of wild type and pcd mice on postnatal day 20, before the appearance of clear-cut phenotypic abnormalities. We identified 300 differentially expressed genes, most of which were involved in metabolic and physiological processes. Among the differentially expressed genes were several calcium binding proteins including calbindin -28k, paravalbumin, matrix gamma-carboxygluta mate protein and synaptotagamins 1 and 13, suggesting the involvement of abnormal Ca2+ signaling in the pcd phenotype.

Expression, purification and characterization of recombinant Jerdonitin, a P-II class snake venom metalloproteinase comprising metalloproteinase and disintegrin domains

Jerdonitin is a P-II class snake venom metalloproteinase comprising metalloproteinase and disintegrin domains. In this study, we established a high-level expression system in Pichia pastoris and developed a purification strategy for the recombinant Jerdonitin. This recombinant Jerdonitin degraded fibrinogen at a level of activity comparable with its wild type. The effects of recombinant Jerdonitin on inhibiting ADP-induced human platelet aggregation were in a dose-dependent manner with an IC50 of 248 nM. In addition, we reported here that Jerdonitin can significantly inhibit the growth of several cell lines, including human liver cancer cells (Bel7402), human leukemia cells (K562) and human gastric carcinoma cells (BGC823). This study offers recombinant Jerdonitin that will be valuable for further functional and structural studies of Jerdonitin. (C) 2009 Elsevier Ltd. All rights reserved.

Rana grylio virus thymidine kinase gene: an early gene of iridovirus encoding for a cytoplasmic protein

The presence of thymidine kinase (TK) is a feature of many large DNA viruses. Here, a TK gene homologue was cloned and characterized from Rana grylio virus (RGV), a member of family Iridoviridae. RGV TK encodes a protein of 195 aa with a predicted molecular mass of 22.1 kDa. Homologues of the protein were present in all the currently sequenced iridoviruses, and phylogenetic analysis showed that it was much close to cellular TK type 2 (TK2), deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Subsequently, Western blotting revealed TK expression increased with time from 6 h post-infection in RGV-infected cells. Using drug inhibition analysis by protein synthesis inhibitor (cycloheximide) and DNA replication inhibitor (cytosine arabinofuranoside), RGV TK was classified as the early expression gene during in vitro infection. Subcellular localization by TK-GFP fusion protein expression and immunofluorescence staining showed RGV TK was an exclusively cytoplasmic protein in fish cells. Collectively, current data indicate that RGV TK was an early gene of iridovirus which encoded a cytoplasmic protein in fish cells.

Cell surface display of functionally active lipases from Yarrowia lipolytica in Pichia pastoris

The lipase genes of Yarrowia lipolytica, LIPY7 and LIPY8, fused with FLO-flocculation domain sequence from Saccharomyces cerevisiae at their N-termini, were expressed in Pichia pastoris KM71. Following the induction with methanol, the recombinant proteins were displayed on the cell surface of P. pastoris, as confirmed by the confocal laser scanning microscopy. The LipY7p and LipY8p were anchored on P. pastoris via the flocculation functional domain of Flo 1 p. The surface-displayed lipases were characterized for their application as the whole-cell biocatalyst. These lipases can also be cleaved off from their anchor by enterokinase treatment to yield functionally active proteins in the supernatant offering an alternative purification method for LipY7p and LipY8p. (c) 2007 Elsevier Inc. All rights reserved.

Molecular and expression characterization of three gonadotropin subunits common alpha, FSH beta and LH beta in groupers

A SMART cDNA plasmid library was constructed from protogyous greasy grouper (Epinephelus coioides) pituitary, and the full-length cDNAs of three gonadotropin (GTH) subunits common alpha, FSH beta and LH beta were cloned and sequenced from the library. The nucleotide sequences of common alpha, FSH beta and LH beta subunit cDNAs are 647, 594 and 574 bp in length, and encode for mature peptides of 94, 99 and 115 aa, respectively. High homology was observed by amino acid sequence alignment and identity comparison of the grouper mature peptides of common alpha, FSH beta and LH beta with that of other fishes. Phylogenetic tree analyses of the three GTH mature subunits revealed similar phylogeny relationships among the studied fish species. Three polyclonal antibodies were prepared from the in vitro expressed common alpha, FSH beta and LH beta mature proteins, respectively. Western blot analysis and immunofluoresence localization were performed on two typical stages of ovarian development stages in red-spotted grouper. Significant differences in protein expression levels of three gonadotropin subunits were revealed between the two ovarian development stages. In the individuals with resting ovary, common alpha was almost not detected in pituitaries, and FSH beta and LH beta expression levels were very low. While in the individuals with developing ovary, the expression of all three gonadotropin subunits reached to a high level. Immunofluoresence localization indicated that the grouper FSH beta cells mainly distributed in the middle area of PPD, while the LH beta cells distributed more widely, including in the area similar to the FSH beta cells and at the external periphery of pituitary near to the PI side. The common alpha might be expressed in both FSH beta and LH beta cells. Double immunofluoresence localization further demonstrated FSH beta and LH beta expression in distinct cells in the PPD area, although the FSH beta and LH beta cells were detected in the identical area of PPD. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

中国沙棘(Hippophae rhamnoides subsp. sinensis)不同种群对干旱胁迫的响应差异与干旱诱导蛋白的分析

沙棘广泛分布于亚欧大陆的温带地区和亚洲亚热带的高海拔地区。沙棘能适应多种生态环境，能耐受多种逆境(如干旱、低温、高温和盐害等)。在中国，沙棘常常被用作植被恢复中的先锋树种而大量栽培。本文以中国沙棘为试验材料，探索沙棘适应干旱机制，以及沙棘对干旱胁迫的适应机制是否存在种群间的差异，同时试图通过分析干旱胁迫下沙棘叶片蛋白质表达变化探索沙棘适应干旱胁迫的分子机理。 对三个分别来自低海拔湿润地区、低海拔干旱地区和高海拔湿润地区的中国沙棘种群进行干旱胁迫处理。干旱胁迫能提高根冠比，比叶面积，降低平均叶面积和总生物量，提高沙棘的抗氧化性酶活性、脯氨酸含量、脱落酸(ABA)含量、降低光合作用，提高长期用水效率。实验中的这两个低海拔种群比高海拔种群抵抗干旱的能力更强,不同的种群采用了不同的策略来耐受干旱胁迫和过氧化胁迫。 在2004 年度的实验中，干旱胁迫处理下，高海拔湿润种群(道孚种群)严重失水，生长也受到更大的抑制，非气孔因素在抑制光合作用方面占支配地位，抗坏血酸含量下降，ABA和脯氨酸含量增加幅度比九寨沟种群的要高，这可能是因为道孚种群严重失水而引起的；而低海拔湿润种群(九寨沟种群)的体内水分状况几乎不受干旱的影响，生长情况也较道孚种群要好。 在2005 年度的试验中，和高海拔湿润地区种群(道孚)相比较，低海拔干旱地区种群(定西)在叶片相对水含量、根冠比、抗氧化酶活性(过氧化氢酶、抗坏血酸过氧化物酶和谷胱甘肽过氧化物酶)、保护性物质(脯氨酸，脱落酸)含量等方面都要高，光能热耗散能力也更强，而且气体交换参数(气孔扩散阻力和胞间CO2浓度等)对干旱也更不敏感。 分析了干旱胁迫下沙棘叶片蛋白质表达的变化。共发现319 个蛋白质，有4 个蛋白在干旱胁迫下消失(Putative ABCtransporter ATP-binding protein 、Hypothetical proteinXP-515578，热激蛋白Hslu219 和一个没得到鉴定的蛋白)，4 个只在干旱胁迫下出现(没命名的蛋白质产物，对甲基苯-丙酮酸双加氧酶，NTrX 和一个没得到鉴定的蛋白)，46 个蛋白质的表达丰度变化显著，包括32 个干旱负调蛋白，14 个干旱正调蛋白(3 个Rubisco 的大亚基、J-type–co-chaperone Hsc20、putative protein DSM3645-2335、putative acyl-COA 脱氢酶、nesprin-2 和两个没有得到鉴定的蛋白质)。这些蛋白质参与了氮代谢调控、抗氧化行物质的合成、脂肪酸β－氧化、核骨架构造、[Fe-S]基团组装、物质跨膜运输、细胞分裂或作为分子伴侣和蛋白质酶起作用。putative ABC transporter ATP-binging protein、NtrX、nesprin-2 和Hslu 是本试验新发现的高等植物蛋白，我们主要从它们的保守结构域或在其他生物中的同源物来猜测它们的功能。实验结果为我们研究植物抗干旱机制提供了新线索和新视野。 Seabuckthorn (Hippophae rhamnoides L.) is widly distributed throughtout the temperatureresiogn of Europe and Asia and sub-tropical plateau zone of Asia. H. rhamnoides can adapatseveral different environments, and can tolerant several envioronmental stresses (e.g, lowtemperature, high temperature, drought and salty). It has been widely used in forest restoration asthe pioneer species in China. In present study, we applied H.rhamnoides subsp. Sinensis asexperimental materials to study its drought-tolerant mechanism, and expected to findpopulational difference in drought-tolerant mechanism that may exist among populations, and tryto get some insight in drought-tolerant mechanism of it at morecular level through analyzing thechange of leaf protein expression. Three populations from high altitude wet zone, low altitude wet zone and low altitude arid znoe,respectively, were applied in our experiment, and were subjected to drought. Drought increasedthe root/shoot ratio(RS), special leaf area, long-term water use efficinency, activity of antioxidantenzymes, proline content and abscisic acid (ABA) content, declined the net photosynthesis rate(A), average leaf area (ALA), total biomass (TB). Both two low altitude populations were moredrought-tolerant than the high altitude population, and different population applied differentstratedgies to tolerant oxidant stress and drought stress. The results of the exprement in 2004 showed that Daofu population was more drought-sensitivethan Jiuzhai population. Under drought conditions, leaf relative water content (RWC) greatlydecreased in Daofu population, but not in Jiuzhai population. The large loss of water in Daofupopulation resulted in a limitation on A mainly caused by non-stomatal factors, severer suppression in growth rate and a significant reduction in ascorbic acid (AsA) content, comparedwith Jiuzhai population. The greater increase in content of ABA and proline in Daofu populationmay be also induced by large loss in water, so that enable plants to cope with sever drought. In the exprement of 2005, drought significantly increased RS, activities of catalase (CAT),peroxidase (POD), glutathione peroxidase (GPX) and ascorbate peroxidase (APX), and alsosignificantly increased ABA and proline contents. On the other hand, compared with Daofupopulation, drought induced larger RS and activities of CAT, GPX and APX, and higher ABAcontent in Dingxi population, whereas gas exchange traits, e.g., stomatal limitation value (LS) andintercellular CO2 concentration (Ci), were less responsive to drought in Dingxi population thanthose in Daofu population. All these factors enable Dingxi population to tolerant drought betterthan Daofu population. The leaf protein profile of seabuchthorn subjected to drought was analyzed. Altogether 319proteins were detected in well-watered sample, four proteins disappeard by drought (putativeABCtransporter ATP-binding protein, hypothetical protein XP-515578, Hslu219and aunidentified protein), four only appeared under drought (a probable nitrogen regulation protein(NtrX), a 4-hydroxyphenylpyruvate dioxygenase , an unnamed protein product and an identified protein), 32 drought down-regulated proteins, and 14 drought up-regulated proteins (nine wereidentified: three large subunits of Rubisco, a hypothetical protein DSM3645-23351, a putativeacyl-COA dehydrogenase, a nesprin-2, a J-type-co-chaperone HSC20 and two unmatchedproteins). These proteins may involve in β-oxidation, cross-membrane transport, cell division,cytoskeleton stabilization, iron-sulfur cluster assembly, nitrogen metabolism regulation andantioxidant substance biosynthesis or function as molecular chaperone or protease. Four proteins(a putative ABC transporter ATP-binging protein, NtrX, nesprin-2, Hslu) were new found in highplants, and their functions were estimated from their conserved domain or their homologues inother organism. Our results provided new clue and new insight for us to study thedrought-tolerant mechanism in plants.

Evaluation of different culture conditions for high-level soluble expression of human cyclin A(2) with pET vector in BL21 (DE3) and spectroscopic characterization of its inclusion body structure

In this paper, we evaluated various parameters of culture condition affecting high-level soluble expression of human cyclin A, in Escherichia coli BL21(DE3), and demonstrated that the highest protein yield was obtained using TB(no glycerol) + 0.5% glucose medium at 25 degrees C. By single immobilized metal ion affinity chromatography, we got highly purified human cyclin A(2) with a yield ranged from 20 to 30 mg/L. By amyloid-diagnostic dye ThT binding and Fourier transform infrared spectroscopy, we observed a significant decrease in alpha-helix content and an increase in beta-sheet structure in cyclin A(2) inclusion body in comparison to its native protein, and confirmed the resemblance of the internal organization of cyclin A(2) inclusion body and amyloid fibrils.

Method for large-scale isolation and purification of R-phycoerythrin from red alga Polysiphonia urceolata Grev

R-phycoerythrin was isolated and purified from a red alga, Polysiphonia urceolata Grev, using Streamline column combined with ion-exchange chromatography or hydroxyapatite chromatography. The purity of R-phycoerythrin isolated by Streamline column was up to 1.66 and the yield of R-phycoerythrin could be as high as 0.68 mg/g frozen P. urceolata. All the eluates from Streamline column were divided into two equivalent parts, respectively. One part was pumped into the ion-exchange column loaded with Q-Sepharose and the other was applied to the adsorption column loaded with hydroxyapatite. The purities of R-phycoerythrin purified using these two methods were both up to 3.26, more than 3.2 the commonly accepted criterion. The yield of purified R-phycoerythrin from the ion-exchange chromatography was 0.40 mg/g frozen P. urceolata and that from the hydroxyapatite chromatography could reach 0.34 mg/g frozen P. urceolata. The purified protein had three absorption peaks at 498, 535, and 565 nm and displayed a fluorescence maximum at 580 nm, which was consistent with the typical spectrum of R-phycoerythrin. The purified R-PE was also identified with electrophoresis. Only one single protein band appeared on native-PAGE with silver staining. SDS-PAGE demonstrated the presence of one 20 kDa major subunit, and one low intensity band corresponding to 33 kDa subunit. The results indicate that using the expanded bed adsorption combined with ion-exchange chromatography or hydroxyapatite chromatography, R-phycoerythrin can be purified from frozen P. urceolata on large scale. (c) 2006 Elsevier Inc. All rights reserved.