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Purification of R-phycoerythrin From Gracilaria Lemaneiformis by Centrifugal Precipitation Chromatography

Purification of R-phycoerythrin From Gracilaria Lemaneiformis by Centrifugal Precipitation Chromatography

Dongyu Gu, Rodrigo Lazo-Portugal, Chen Fang, Zhantong Wang, Ying Ma, Martha Knight, Yoichiro Ito

J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Jun 15;1087-1088:138-141.

PMID: 29738963

Abstract:

Centrifugal precipitation chromatography (CpC) is a powerful chromatographic technique invented in the year 2000 but so far very little applied. The method combines dialysis, counter-current and salting out processes. The separation rotor consists of two identical spiral channels separated by a dialysis membrane (6-8 K MW cut-off) in which the upper channel is eluted with an ammonium sulfate gradient and the lower channel with water, and the mixtures are separated according to their solubility in ammonium sulfate as a chromatographic technique. In the present study, the method was successfully applied for separation and purification of R-phycoerythrin (R-PE), a protein widely used as a fluorescent probe, from the red alga Gracilaria lemaneiformis. The separation was performed with the elution of ammonium sulfate from 50% to 0% in 21.5 h at a flow rate of 0.5 ml/min, while the lower channel was eluted with water at a flow rate of 0.05 ml/min after sample charge, and the column was rotated at 200 rpm. After a single run, the absorbance ratio A565/A280 (a criterion for the purity of R-PE) was increased from 0.5 of the crude to 6.5. The purified R-PE exhibited a typical "three peaks" spectrum with absorbance maximum at 497, 538 and 565 nm. The Native-PAGE showed one single protein band and 20 kDa (subunits α and β) and 30 kDa (subunit γ) can be observed in SDS-PAGE analysis which were consistent with the (αβ)6γ subunit composition of R-PE. The results indicated that CpC is an efficient method to obtain protein with the high purity from a complex source.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP11016174	R-Phycoerythrin		11016-17-4	Price

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