One-step chromatographic procedure for purification of B-phycoerythrin from Porphyridium cruentum

Highlights

• Osmotic shock and ultrafiltration were employed for extraction and initially purified purification of B-PE.

• Futher purification was carried out using only a one-step SOURCE 15Q chromatography.

• Analytical grade B-PE was obtained with a purity ratio (A₅₄₅/A₂₈₀) of 5.1 and a yield of 68.5%.

• The absorption spectrum, fluorescence spectrum, and the subunits molecular weights of purified B-PE were analyzed.

Abstract

B-phycoerythrin (B-PE) was separated and purified from microalga Porphyridium cruentum using one-step chromatographic method. Phycobiliproteins in P. cruentum was extracted by osmotic shock and initially purified by ultrafiltration. Further purification was carried out with a SOURCE 15Q exchange column and analytical grade B-PE was obtained with a purity ratio (A₅₄₅/A₂₈₀) of 5.1 and a yield of 68.5%. It showed a double absorption peaks at 545 nm and 565 nm and a shoulder peak at 498 nm, and displayed a fluorescence emission maximum at 580 nm. The analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) showed a bulky band between 18 and 20 kDa which could be assigned to subunits α and β and a low intensity band of 27 kDa assigned to γ subunit. Our protocol provides attractive alternative to consider for the purification procedure to obtain analytical grade B-PE at commercial level.

Introduction

Phycobiliproteins (PBPs) are a class of chromoproteins found in red algae, cyanobacteria, cryptophytes, and glaucocystophytes [1], [2], [3], [4], [5]. According to their spectral properties, PBPs are commonly classified phycoerythrins (PE, λ_max = 540–570 nm), phycoerythrocyanins (PEC, λ_max = 560–600 nm), phycocyanins (PC, λ_max = 610–620 nm), and allophycocyanins (APC, λ_max = 650–655 nm) [6], [7]. These proteins are an ensemble of photosynthetic complex called phycobilisome and work as accessory pigments to allow light absorption in the cell on the thylakoids of chloroplast [8], [9], [10].

Depending on their absorption spectrum, PE can be divided into three main classes R-PE, B-PE, and C-PE [11]. It has been reported that PE is useful for development of fluorescent probes because of its high extinction coefficients, fluorescence quantum efficiency of more than 0.8, and stability as hexamers even at lower concentrations without showing fluorescence decrease [12], [13], [14], [15]. Moreover, PE can be used as natural colorants in the food, cosmetic industry and as photo sensitizers in tumor photodynamic therapy [16], [17], [18], [19], [20], [21], [22], [23]. In case of PE, commercial scale processing of this material continues to be a daunting task. R-PE is usually extracted and purified from Porphyra, Grateloupia and Polysiphonia, but most of them contained huge amount of polysaccharides which form gel (carbohydrate) and thereby making the purification task most difficult and tedious [2], [24], [25], [26], [27]. B-PE is the main phycobiliprotein of Porphyridium species and it contains three subunits (αβ)₆γ with molecular weights of 19.5 kDa (α and β subunits) and 29 kDa (γ subunit) for a total molecular weight of 263 kDa [10]. This phycoerythrin normally has a double absorption peaks at ∼545 and ∼565 nm, and a shoulder peak at ∼498 nm, and a fluorescence emission maximum at 580 nm [11]. The price of B-PE products varies widely and is dependent on the purity ratio (A_max/A₂₈₀). The cost of analytical grade B-PE (purity ratio higher than 4.0) can be as high as about 50 US$/mg [22]. This high value makes attractive attempt to develop an efficient purification procedure to obtain B-PE with high purity and yield at commercial level. Previous attempt for analytical grade of B-PE has been reported [9], [11], [28], [29], [30]. However most cases are complicated by the need for multiple chromatography steps to obtain highly purified protein. The complexity of the procedures for purification of B-PE is usually characterized for having a high number of stages, low yield and limitation to easy process scale-up, which limits its potential practical implementation at commercial level [22]. Therefore, it is needed to develop an efficient purification procedure to obtain analytical grade of B-PE with high yield at large scale. SOURCE 15Q is an ion exchanger of high performance separation medium for fast, preparative purification of biomolecules. It is ideal for intermediate purification steps in industrial processes where high productivity and maintained performance at large scale are important. In the present study, we have improved the efficiency of purification step of the routine methods to obtain B-PE from P. cruentum, involving only a one-step chromatography on a SOURCE 15Q ion exchanger column.