Ruperto Bermejo

Preparative purification of B-phycoerythrin from the microalga Porphyridium cruentum by expanded-bed adsorption chromatography

B-Phycoerythrin (B-PE) is a major light-harvesting pigment of microalgae. Due to its high fluorescence efficiency and its intense and unique pink color, it is widely used as a fluorescent probe and analytical reagent as well as being employed as a natural dye in foods and cosmetics. Tedious methodologies for B-PE purification have been published. In this work we present a new, fast, preparative and scaleable two-step chromatographic method for B-PE purification from the red microalga Porphyridium cruentum. Initially, phycobiliproteins were released from the microalga cells by osmotic shock and captured by applying the centrifuged cell suspension to a column containing 74 ml Streamline-DEAE equilibrated with 50 mM acetic acid-sodium acetate buffer, pH 5.5, using expanded-bed adsorption chromatography at an upward flow of 200 cm h(-1). After adsorption, washing was carried out in the expanded-bed mode. Having removed unbound proteins and cellular debris, the bed was allowed to sediment and a B-PE-rich solution was eluted with a downward flow of the same 250 mM buffer. In order to obtain pure B-PE, we utilized conventional ion-exchange chromatography with a column of DEAE-cellulose loaded directly with the eluate from Streamline-DEAE and developed using a discontinuous gradient of acetic acid-sodium acetate buffer, pH 5.5. With this new methodology, 66% of B-PE contained in the biomass of the microalgae was recovered, a value significantly higher than those obtained following other methodologies. The B-PE purity was tested using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and spectroscopic characterization.

Chromatographic purification and characterization of B-phycoerythrin from Porphyridium cruentum: Semipreparative high-performance liquid chromatographic separation and characterization of its subunits☆

A fast preparative two-step chromatographic method for purification of B-phycoerythrin from Porphyridium cruentum is described. This biliprotein was homogeneous as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielding three closely migrating bands corresponding to its three subunits. Baseline separation of its alpha-, beta- and gamma-subunits was achieved by a reversed-phase HPLC gradient semipreparative method with a C4 large-pore column and a solvent system consisting of 0.05% trifluoroacetic acid (TFA) in water and 0.05% TFA in acetonitrile. B-Phycoerythrin in different aggregation states and its subunits have been spectroscopically characterized. Hexameric B-phycoerythrin has similar secondary and tertiary structure than dissociated B-phycoerythrin determined by circular dichroism.

Chromatographic purification of biliproteins from Spirulina platensis high-performance liquid chromatographic separation of their α and β subunits

A fast preparative two-step chromatographic method for purification of C-phycocyanin and allophycocyanin from Spirulina platensis is described. Both biliproteins were homogeneous as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielding two closely migrating bands. Separation of α and β subunits from C-phycocyanin and allophycocyanin was also accomplished by a chromatographic procedure under denaturing conditions. The phycocyanobilin chromophore from both biliproteins has been isolated, tested for purity by reversed-phase high performance liquid chromatography and spectroscopically characterized.

Development of a process for large-scale purification of C-phycocyanin from Synechocystis aquatilis using expanded bed adsorption chromatography

In this paper a large and scaleable method for purification of C-phycocyanin (C-PC) from the cyanobacteria Synechocystis aquatilis has been developed. Phycobiliproteins are extracted from the cells by osmotic shock and separated by passing the centrifuged cell suspension through an expanded bed adsorption chromatography (EBAC) column using Streamline-DEAE as adsorbent. The eluted C-PC rich solution is finally purified by packed-bed chromatography using DEAE-cellulose. Optimal extraction is achieved using phosphate 0.05 M buffer pH 7.0 twice. The operation of EBAC is optimized on a small scale using a column of 15 mm internal diameter (I.D.). The optimal conditions are a sample load of 4.9 mg C-PC/mL adsorbent, an expanded bed volume twice the settled bed volume and a sample viscosity of 1.020 mP. The EBAC process is then scaled up by increasing the column I.D. (15, 25, 40, 60 and 90 mm) and the success of the scale-up process is verified by determining the protein breakthrough capacity and product recovery. The yield of the EBAC step is in the range of 90-93% for every column diameter. To obtain pure C-PC, conventional ion-exchange chromatography with DEAE-cellulose is utilized and a yield of 74% is obtained. The overall yield of the process, comprising all steps, is 69%. The purification steps are monitored using SDS-PAGE and the purity of recovered C-PC is confirmed by absorption and emission spectroscopy and RP-HPLC. Results show that EBAC method is a scalable technology that allows large quantities of C-PC to be obtained without product loss, maintaining a high protein recovery while reducing both processing cost and time.

Fluorescent behavior of B-phycoerythrin in microemulsions of aerosol OT/water/isooctane.

Taking advantage of its unusual fluorescent properties, the incorporation of B-phycoerythrin (B-PE) in aerosol OT (AOT, sodium bis-(2-ethylhexyl) sulphosuccinate)/water/isooctane microemulsions was investigated by following their steady-state and time-resolved fluorescence as a function of the water-to-surfactant molar ratio, w(0). The fluorescent intensity at 575 nm increased continuously with increasing water content, saturating at a w(0) around 35 and staying practically constant at w(0)> or =40. The steady-state anisotropy showed an initial increase with increasing water content until w(0)=23 and then decreased strongly, staying practically constant when w(0)> or =40. The values of the fluorescent parameters, anisotropy and fluorescent intensity, were unchanged when the water content of the system increased in the range between w(0)=40 to 50. This implies the effective incorporation of B-PE in the microemulsion droplets with w(0)> or =40, as well as the equilibrium of the dispersion at these water/surfactant ratios, since higher water content does not affect the main surrounding microenvironment of the protein. The overall incorporation in the microemulsion droplets caused minor spectroscopic changes with respect to biliprotein in aqueous solution of 20 mM sodium phosphate buffer, pH 7.0, such as a blue absorption shift of 3 nm and an emission shift of 1.5 nm, as well as a slight increase in excitation anisotropy spectrum mainly caused by a decrease in protein mobility. Therefore, there are no important interactions between the chromophores and the AOT sulfonate head groups. Emission intensity decays followed complex kinetics in both aqueous and dispersion media. The stability with time and temperature of the biliprotein in the microemulsion was higher than in the aqueous solution. All the results can be explained in terms of B-PE inclusion in the water droplets of AOT microemulsions where the protein has similar configuration and conformation to that in aqueous solution but with the chromophores more protected.

Fluorescein-Labeled DNA Probes for Homogeneous Hybridization Assays: Application to DNA E. Coli Renaturation

The sensitivity of the emission intensity of fluorescein to environmental changes opens the possibility for developing homogeneous DNA assays. In this research, we were able to quickly and easily detect nucleic acid renaturation by using an E. coli DNA with approximately 4% conjugation of fluorescein to the N4-aminoethylmodifled cytosine residues. We determined how the emission intensity of fluorescein attached to single-stranded DNA decreases when renatured to double-stranded DNA. The decrease in fluorescence efficiency is due to changes in the fluorescein monoanion–dianion ratio because of pH modifications in the surrounding of the fluorescein label. The decrease in fluorescence intensity was used for analysis of DNA E. coli renaturation kinetics.

C-phycocyanin incorporated into reverse micelles: a fluorescence study

The solubilization of C-PC into aerosol-OT(AOT, sodium bis(2-ethylhexyl)sulphosuccinate):water:i-octane reverse micelles has been investigated by fluorescence spectroscopy by following its behaviour as a function of the water-to-surfactant molar ratio, w0. The maximum wavelength emission decreased steadily with an increase in water content, together with a concomitant increase in steady-state anisotropy. These effects saturated at a w0 of around 30 and remained practically constant at w0\30. These results are explained in terms of an inclusion into the inner core of the reverse AOT micelles. Strong interactions also took place between amino-acid residues neighbouring bilin chromophores and the AOT sulfonate headgroups, which resulted in the chromophores being located in the structured interfacial micellar water layer. The stabilization rate of the spectral parameters from micelles of protein solutions at pH 5.0 followed pseudo-first-order kinetics and afterwards remained stable for some days. The fluorescence intensity decay of C-PC in reverse micelles is described by a triple exponential function, showing a similar complex pattern to that in an aqueous solution. The fluorescence lifetime values of micelled C-PC indicate that the chromophores are shielded against solvent quenching by the structured interfacial water layer.

Labeling of cytosine residues with biliproteins for use as fluorescent DNA probes

Abstract The fluorescent properties of biliproteins (B-phycoerythrin, BPE; C-phycocyanin, CPC and allophycocyanin, APC) have been utilized as labels of nucleic acids to detect hybridization by means of steady-state fluorescence anisotropy in a homogeneous aqueous solution of model system in which poly(C) and poly(I) are, respectively, the probe and target sequences. An easy method to obtain biliprotein–DNA conjugates by a two-stage procedure is described. The first stage was a modification of the cytosine amino group of poly(C) at the N 4 position which then reacted with N -succinimidyl 3-(2-pyridyldithio) propionate (SPDP) to obtain poly(C)-bound 2-pyridyl disulfide. In the second stage biliproteins were directly reacted with SPDP to obtain biliprotein-bound 2-pyridyl disulfide, dithiotreitol was added for reduction to biliprotein-bound thiol and was then mixed with the poly(C)-bound 2-pyridyl disulfide to obtain the biliprotein–poly(C) conjugate. The three biliproteins studied bind to nucleic acids without noticeable change of their spectral properties (absorption, fluorescence efficiency and fluorescence lifetime). Consequently, our labeling methodology can be applied to obtain any type of biliprotein-labeled nucleic acid probe. The increase of the steady-state fluorescence anisotropy from biliprotein–poly(C) upon hybridization with poly(I) can be used to readily detect the hybridization with the target poly(I) in a sample without having to separate free and bound labeled probes. The small decreases in lifetime displayed by the biliprotein–poly(C) conjugates upon hybridization are not sufficient to explain the steady-state anisotropy increase. Apparently, the rotational motion of the overall macrostructure is principally responsible for the increase in anisotropy. The greater fluorescence efficiency and lifetime from BPE with respect to those from CPC or APC allows us recommend that protein as the most suitable label.

Pilot-Scale Recovery of Phycoerythrin from Porphyridium cruentum using Expanded Bed Adsorption Chromatography

In this paper, a process was developed on a pilot scale for the recovery of B-phycoerythrin from Porphyridium cruentum using EBA. The process was optimized on a small scale using a 15 mm ID column, then scaled up to 150 mm ID columns. In the developed process, the phycobiliproteins were extracted by osmotic shock and then separated by applying the centrifuged cell suspension to an EBA column. Following this, the PE-rich solution was eluted to final purity using packed bed chromatography. Optimal conditions to separate B-phycoerithrin using EBA on a small scale were: 0.88 mg B-PE/mL adsorbent sample load, H/H0 = 2 and a sample viscosity of 1.068 mP. The EBA process was then scaled up by increasing the ID column (15, 25, 40, 60, and 90 mm) and finally the process was developed on a pilot scale using a 150 mm ID column (a scale-up factor of 100). The yield from the EBA step ranged between 71–78%, whichever ID column was used. The overall purification process yield was 54%, purification steps were monitored using SDS-PAGE, whereas protein purity was confirmed using spectroscopy. Results show that EBA is a scalable technology that allows large quantities of B-PE to be obtained, thus reducing product loss and maintaining high protein recovery while reducing both processing cost and time.

Fluorescence energy transfer between fluorescein label and DNA intercalators to detect nucleic acids hybridization in homogeneous media

A general approach to detecting nucleic acid sequences in homogeneous media by means of steady-state fluorescence measurements is proposed. The methodology combines the use of a fluorescence-labeled single-strand DNA model probe, the complementary single-strand DNA target, and a DNA intercalator. The probe was fluorescein labeled to a spacer arm at the N4 position of the cytosine amino groups in polyribocytidylic acid (5′), poly(C), which acts as a model DNA probe. The complementary strand was polyriboinosinic acid (5′), poly(I), as a model of the target, and the energy transfer acceptor was an intercalator, either ethidium bromide or ethidium homodimer. In previous papers we have shown that the fluorescence intensity of the fluorescein label decreases when labeled poly(C) hybridizes with poly(I), and this fluorescence quenching can be used to detect DNA hybridization or renaturation in homogeneous media. In this paper we demonstrate that fluorescence resonance energy transfer (FRET) between fluorescein labeled to poly(C) and an intercalator agent takes place when single-stranded poly(C) hybridizes with poly(I), and we show how the fluorescence energy transfer further decreases the steady-state fluorescence intensity of the label, thus increasing the detection limit of the method. The main aim of this work was to develop a truly homogeneous detection system for specific nucleic acid hybridization in solution using steady-state fluorescence and FRET, but with the advantage of only having to label the probe with the energy donor since the energy acceptor is intercalated spontaneously. Moreover, the site label is not critical and can be labeled randomly in the DNA strand. Thus, the method is simpler than those published previously based on FRET. The experiments were carried out in both direct and competitive formats.