One-step chromatography method for efficient separation and purification of R-phycoerythrin from Polysiphonia urceolata

Phycoerythrins have been widely used in food, cosmetics., immunodiagnostics and analytical reagents. An efficient one-step chromatography method for purification of R-phycoerythrins from Polysiphonia urceolata was described in this paper. Pure R-phycoerythrin was obtained with an absorbance ratio A(565)/A(280) of 5.6 and a high recovery yield of 67-33%, using a DEAE-Sepharose Fast Flow chromatography with a gradient elution of pH, alternative to common gradient elution of ionic strength. The absorption spectrum of R-phycoerythrin was characterized with three absorbance maxima at 565, 539 and 498 mum, respectively and the fluorescence emission spectrum at room temperature was measured to be 580nm. The results of native-PAGE. and SDS-PAGE showed no contamination by other proteins in the phycoerythrin solution. which suggests an efficient method for the separation and purification of R-phycoerythrins from Polysiphonia urceolata. (C) 2004 Elsevier B.V. All rights reserved.

Formation and growth of Bryopsis hypnoides lamouroux regenerated from its protoplasts

Tissue culture, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and spectra analysis were used for studying the aggregation mechanism of protoplasts from Bryopsis hypnoides Lamouroux and the discrepancy between the protoplast-regenerated plants and the wild type. The aggregation of protoplasts from B. hypnoides was observed in natural seawater and artificial seawater with different pH values, and the location and mechanism of the materials causing the aggregation were also studied. Results showed that the protoplasts could aggregate into some viable spheres in natural seawater and subsequently grow into mature individuals. Aggregation of the protoplasts depended exclusively upon the pH value (6-11), and the protoplasts aggregated best at pH 8-9. Some of the extruded protoplasts were separated into two parts by centrifugation: the pellet (PO) and the supernatant (PL). The PO could aggregate in artificial seawater (pH 8.3) but not in PL. No aggregation was found in PO cultured in natural seawater containing nigericin, which can dissipate the proton gradients across the membrane. These experiments suggest that the aggregation of protoplasts is proton-gradient dependent and the materials causing the aggregation were not in the vacuolar sap, but located on the surface or inside the organelles. Furthermore, the transfer of the materials across the membrane was similar to Delta pH-based translocation (Delta pH/TAT) pathway that occurs in the chloroplasts of higher plants and bacteria. Obvious discrepancies in both the total soluble proteins and the ratio of chlorophyll a to chlorophyll b between the regenerated B. hypnoides and the wild type were found, which may be related to the exchange of genetic material during aggregation of the organelles. In the process of development, diatom Amphora coffeaeformis Agardh attached to the protoplast aggregations, retarding their further development, and once they were removed, the aggregations immediately germinated, which showed that diatoms can affect the development of other algae.

Isolation of the main light-harvesting chlorophyll a/b-protein complex from thylakoid membranes of marine alga, Bryopsis corticulans by a direct method

The main chlorophyll a/b light-harvesting complex (LHC 11) has been isolated directly from thylakoid membranes of marine green alga (Bryopsis corticulans Setch.) by two consecutive runs of anion exchange and gel-filtration chromatography. LHC 11 proteins in the membrane extracts treated with 3% n-Octyl-b-D-glucopyranoside (OG) obtained specific binding ability on Q Sepharose column, and thus were isolated from the thylakoid membranes in a highly selective fraction. The monomeric, trimeric and oligomeric subcomplexes of LHC 11 have been obtained by fractionation of the LHC 11 mixes with sucrose density gradient ultracentrifugation. The SDS-PAGE analysis of peptide composition and absorption spectrum showed that LHC 11 monomers, trimers and oligomers prepared through this work were intact and in high purity. Our report is the first to show that it is possible to purify LHC If directly from thylakoid membranes without extensively biochemical purification.

Isolation and characterization of photosystem II of Porphyra yezoensis ueda

The thylakoid membranes were isolated and purified from gametophyte of Porphyrayezoensis Ueda (P yezoensis) by sucrose density gradient ultracentrifugation. After R yezoensis gametophyte thylakoid membranes were solubilized with SDS, the photosystem 11 (PSII) particles were isolated and purified. The activity of PSII particles was determined with DCIP (2,6-dichloroindophenol) photoreduction reaction. The composition of purified PSII particles was detected by SDS-PAGE. As a result, seven proteins including 55 kD protein, 47 kD protein, 43 kD protein, 33 kD protein, 31 kD protein, 29 kD protein, and 18 kD protein were found. Compared with PSII particles of higher plants and other algae, they were identified as D1/D2 complex, CP47, CP43, 33 kD protein, D1, D2 and cyt c-550 respectively. Besides, other three new proteins of 20 kD, 16 kD and 14 kD respectively were found. Among these extrinsic proteins, the 16 kD and 14 kD proteins had not been reported previously, and the 20 kD protein was found for the first time in multicellular red algae.

Purification and partial characterization of Mn superoxide dismutase from muscle tissue of the shrimp Macrobrachium nipponense

Superoxide dismutase (SOD; EC 1.15.1.1) is an enzyme that protects against oxidative stress from superoxide radicals in living cells. This enzyme had been isolated, purified and partially characterized from muscle tissue of the shrimp Macrobrachium nipponense. The purification was achieved by heat treatment, ammonium sulfate fractionated precipitation and column chromatograph on DEAE-cellulose 32. Some physiological and biochemical characterization of it was tested. The molecular weight of it was about 21.7 kDa, as judged by SDS-polyacrylamide gel electrophoresis. The purified enzyme had an absorption peak of 278 nm in ultraviolet region, and the enzyme remained stable at 25-45 degreesC within 90 min. However, it was rapidly inactivated at higher temperature. Treatment of the enzyme with 1 mM ZnCl2, SDS and 1 mM or 10 mM mercaptoethanol showed some increasing activity. However, the enzyme activity was obviously inhibited by 10 mM CaCl2, CuSO4, ZnCl2 and 1 mM CaCl2 and 10 mM K2Cr2O7. SOD activity did not show significantly variation after incubated with 1 mM CaCl2, EDTA and 10 MM SDS. The enzyme was insensitive to cyanide and contained 1.03 +/- 0.14 atoms of manganese per subunit shown in atomic absorption spectroscopy, which revealed that purified SOD was Mn superoxide dismutase. (C) 2004 Elsevier B.V. All rights reserved.

Structural analysis of peptides of PSII light-harvesting complexes in siphonous green algae, Codium fragile

peptide composition and arrangement of 4 major light-harvesting complexes LHCP1-3 and LHCP3, isolated from siphonous green algae (Codium fragile (Sur.) Hariot.) were investigated. LHCP1 showed five main peptides, 34.4, 31.5, 29.5, 28.2 and 26.5 kD in SDS-PAGE, the 34.4 and 31.5 kD peptides were never found in higher plants. LHCP3 contained the other four kinds of LHCP1 peptides except 34.4 kD, while LHCP3, consisted of only 28.2 and 26.5 kD peptides. We found that 34.4, 28.2 and 26.5 kD peptides were easy to decompose from LHCP1 when subjected to SDS-PACE without pretreatment. They might be located at the exterior of LHCP1, while the 31.5 and 29.5 kD peptides were at the central part. The 28.2 and 26.5 kD peptides often occurred in CPa, the center complex of PS II. They are possibly the LHC II peptides tightly associated with CC II. According to the results described above, a peptide map of LHCP1 was sketched.

Isolation of PSI from a siphonous marine green alga, Codium fragile

Three different forms of PS I complexes were isolated from a siphonous marine green alga, Codium fragile, by Triton X-100 sucrose gradient centrifugation. Zone III had a Chl a/b>20, and designated as PS I. core complex CC I because it created only CP I band in mild PAGE. Zone IV and V had absorption at 436 and 674 nm, 467 and 650 nm, and 540 nm, suggesting the presence of Chl a, Chl b, siphonaxanthin and siphonein, Chl a/b were 3.23 and 2.4, respectively. Both CP I and CP I a bands were observed when they were subjected to mild PAGE. Therefore, Zone IV and V were different forms of PS I complexes that consisted of CC I and different amount of light-harvesting complex LHC I. Zone III contained only 66 and 56 ku peptides in SDS-PAGE, while Zone IV and V had 4 different LHC I peptides of 25, 26, 26.2 and 27.5 ku in addition to 66, 56 ku peptides. Fluorescence emission spectra showed that efficient energy transfer were kept among pigments in isolated PS I complexes. Excitation energy absorbed by Chl b, siphonaxanthin and siphonein can be transferred to Chl a.

Expression of TNF-alpha gene in Anabaena sp PCC 7120 and purification of its product by affinity chromatography

The effects of N (NaNO3) and C (NaAc) source in medium on the expression of tumor necrosis factor-alpha (TNF-alpha) gene in transgenic Anabaena sp. PCC 7120 were compared. The data showed that N source stabilized the expression of foreign protein and C source altered the synthesis of cell walls. Comparing several methods for breaking the cells, supersonic was able to extract TNF-alpha better than others. For purification of TNF-alpha, transgenic Anabaena cells were broken, the extracts were precipitated with ammonia sulfate, and the impure TNF-alpha was eluted from DEAE ion exchange chromatography. Electrophoresis (PAGE-SDS) showed a single band at 17 kD position.

The complete amino acid sequence of growth hormone and partial amino acid sequence of prolactin and somatolactin from sea perch (Lateolabrax japonicus)

Growth hormone (GH), prolactin (PRL) and somatolactin (SL) were purified simultaneously under alkaline condition (pH 9.0) from pituitary glands of sea perch (Lateolabrax japonicas) by a two-step procedure involving gel filtration on Sephadex G-100 and reverse-phase high-performance liquid chromatography (rpHPLC). At each step of purification, fractions were monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting with chum salmon GH. PRL and SL antisera. The yields of sea perch GH, PRL and SL were 4.2, 1.0 and 0.28 mg/g wet tissue, respectively. The molecular weights of 19,200 and 20,370 Da were estimated by SDS-PAGE for sea perch GH and PRL, respectively. Two forms of sea perch SL were found: one (28,400 Da) is probably glycosylated, while the other one (23,200 Da) is believed to be deglycosylated. GH bioactivity was examined by an in vivo assay. Intraperitoneal injection of sea perch GH at a dose of 0.01 and 0.1 mug/g body weight at 7-day intervals resulted in a significant increase in body weight and length of juvenile rainbow trout. The complete sea-perch GH amino acid sequence of 187 residues was determined by sequencing fragments cleaved by chemicals and enzymes. Alignment of sea-perch GH with those of other fish GHs revealed that sea-perch GH is most similar to advanced marine fish, such as tuna, gilthead sea bream, yellowfin porgy, red sea bream, bonito and yellow tail with 98.4, 96.2%, 95.7%, 95.2%, 94.1% and 91% sequence identity, respectively. Sea-perch GH has low identity to Atlantic cod (76.5%), hardtail (73.3%), flounder (68.4%), chum salmon (66.3%), carp (54%) and blue shark (38%). Partial amino-acid sequences of 127 of sea-perch PRL and the N-terminal of 16 amino-acid sequence of sea-perch SL have been determined. The data show that sea-perch PRL has a slightly higher sequence identity with tilapia PRL( 73.2%) than with chum salmon PRL(70%) in this 127 amino-acid sequence. (C) 2001 Elsevier Science B.V. All rights reserved.

Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium, Anabaena sp. PCC 7120

The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDC-TNF. The expression of the rhTNF gene in Escherichia coil has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced into Anabaena sg PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the introduced plasmid pDC-TNF in recombinant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis with the extracted plasmid samples and Southern blotting with alpha-(32)p labeled hTNF cDNA probes, while the expression of the hTNF gene in Anabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cytotoxicity of the TNF in the crude extracts from the transgenic cyanobacterium Anabaena sp. PCC 7120.

Purification and characterization of agarases from a marine bacterium Vibrio sp. F-6

Marine bacterium Vibrio sp. F-6, utilizing agarose as a carbon source to produce agarases, was isolated from seawater samples taken from Qingdao, China. Two agarases (AG-a and AG-b) were purified to a homogeneity from the cultural supernatant of Vibrio sp. F-6 through ammonium sulfate precipitation, Q-Sepharose FF chromatography, and Sephacryl S-100 gel filtration. Molecular weights of agarases were estimated to be 54.0 kDa (AG-a) and 34.5 kDa (AG-b) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH values for AG-a and AG-b were about 7.0 and 9.0, respectively. AG-a was stable in the pH range of 4.0-9.0 and AG-b was stable in the pH range of 4.0-10.0. The optimum temperatures of AG-a and AG-b were 40 and 55 degrees C, respectively. AG-a was stable at temperature below 50 degrees C. AG-b was stable at temperature below 60 degrees C. Zn2+, Mg2+ or Ca2+ increased AG-a activity, while Mn2+, Cu2+ or Ca2+ increased AG-b activity. However, Ag+, Hg2+, Fe3+, EDTA and SDS inhibited AG-a and AG-b activities. The main hydrolysates of agarose by AG-a were neoagarotetraose and neoagarohexaose. The main hydrolysates of agarose by AG-b were neoagarooctaose and neoagarohexaose. When the mixture of AG-a and AG-b were used, agarose was mainly degraded into neoagarobiose.

Isolation and characterization of photosystem II from the filamentous sporophyte of Porphyra yezoensis

Thylakoid membranes were isolated and purified from diploid filamentous sporophytes of Porphyra yezoensis Ueda using sucrose density gradient ultracentrifugation (SDGUC). After thylakoid membranes were solubilized with SDS, the photosystem II (PSII) particles with high 2, 6-dichloroindophenol (DCIP) photoreduction activity were isolated by SDGUC. The absorption and fluorescence spectra, DCIP photoreduction activity and oxygen evolution activity of the thylakoid membranes and PSII particles were determined. The polypeptide composition of purified PSII particles was distinguished by SDS-PAGE. Results showed that PSII particles of sporophytes differed from the gametophytes in spectral properties and polypeptide composition. Apart from 55 kDa D1-D2 heterodimer, CP47, CP43, 33 kDa protein was also detected. However, cyt c-550, 20 kDa, 14 kDa and 16 kDa proteins found in PSII particles from gametophytes were not detected in the sporophytes.

Induction and characterization of green pigmentation mutant in Porphyra yezoensis Ueda

The free living conchocelis of Porphyra yezoensis Ueda was treated with N-methyl-N-nitro-N-nitrosoguanidine to induce pigmentation mutants. The artificial green pigmentation mutant of P. yezoensis conchocelis, which was composed entirely of green cells, was isolated through visualization with the unaided eye. The acquired green conchocelis was further developed into a green gametophytic blade. This mutant was relatively stable in color in both gametophytic blade and conchocelis phases. The gametophytic blade mutant was successively cultivated for commerce at some Porphyra farms in Rudong, China, and few wild type or sectorially variegated gametophytic blade occurred, indicating that the green mutant has commercial value. The green mutant was characterized as having lower phycoerythrin and higher phycocyanin content, and SDS-PAGE suggested that phycoerythrin was missing the gamma-subunit in comparison to the wild type. The wild type and the green mutant showed a clear difference in 02 evolution rates in white, green, yellow, and red light, which might be due to the qualitative and quantitative changes of phycoerythrin, and the quantitative difference of phycocyanin. (C) 2008 Elsevier B.V. All rights reserved.

Large-scale recovery of C-phycocyanin from Spirulina platensis using expanded bed adsorption chromatography

C-phycocyanin was purified on a large scale by a combination of expanded bed adsorption, anion-exchange chromatography and hydroxyapatite chromatography from inferior Spirulina platensis that cannot be used for human consumption. First, phycobiliproteins were extracted by a simple, scaleable method and then were recovered by Phenyl-Sepharose chromatography in an expanded bed column. The purity (the A(620)/A(280) ratio) of C-phycocyanin isolated with STREAMLINE (TM) Column was up to 2.87, and the yield was as high as 31 mg/g of dried S. platensis. After the first step, we used conventional anion-exchange chromatography for the purification steps, with a yield of 7.7 mg/g of dried S. platensis at a purity greater than 3.2 and with an A(620)/A(650) index higher than 5.0. The fractions from anion-exchange chromatography with a level of purity that did not conform to the above standard were subjected to hydroxyapatite chromatography, with a C-PC yield of 4.45 mg/g of dried S. platensis with a purity greater than 3.2. The protein from both purification methods showed one absolute absorption peak at 620 nm and a fluorescence maximum at 650 nm, which is consistent with the typical spectrum of C-phycocyanin. SDS-PAGE gave two bands corresponding to 21 and 18 kDa. In-gel digestion and LC-ESI-MS showed that the protein is C-phycocyanin. (c) 2006 Elsevier B.V. All rights reserved.

Axial growth of hexactinellid spicules: Formation of cone-like structural units in the giant basal spicules of the hexactinellid Monorhaphis

The glass sponge Monorhaphis chuni (Porifera: Hexactinellida) forms the largest bio-silica structures on Earth; their giant basal spicules reach sizes of up to 3 m and diameters of 8.5 mm. Previously, it had been shown that the thickness growth proceeds by appositional layering of individual lamellae; however, the mechanism for the longitudinal growth remained unstudied. Now we show, that the surface of the spicules have towards the tip serrated relief structures that are consistent in size and form with the protrusions on the surface of the spicules. These protrusions fit into the collagen net that surrounds the spicules. The widths of the individual lamellae do not show a pronounced size tendency. The apical elongation of the spicule proceeds by piling up cone-like structural units formed from silica. As a support of the assumption that in the extracellular space silicatein(-like) molecules exist that associate with the external surface of the respective spicule immunogold electron microscopic analyses were performed. With the primmorph system from Suberites domuncula we show that silicatein(-like) molecules assemble as string- and net-like arrangements around the spicules. At their tips the silicatein(-like) molecules are initially stacked and at a later stay also organized into net-like structures. Silicatein(-like) molecules have been extracted from the giant basal spicule of Monorhaphis. Applying the SDS-PAGE technique it could be shown that silicatein molecules associate to dimers and trimers. Higher complexes (filaments) are formed from silicatein(-like) molecules, as can be visualized by electron microscopy (SEM). In the presence of ortho-silicate these filaments become covered with 30-60 nm long small rod-like/cuboid particles of silica. From these data we conclude that the apical elongation of the spicules of Monorhaphis proceeds by piling up cone-like silica structural units, whose synthesis is mediated by silicatein(-like) molecules. (C) 2008 Elsevier Inc. All rights reserved.