Facile isolation of purple membrane from Halobacterium salinarum via aqueous-two-phase system

• Published in: Protein expression and purification Vol. 89; no. 2; pp. 219 - 224
• Main Authors: Shiu, Pei-Jing, Ju, Yi-Hsu, Chen, Hsiu-Mei, Lee, Cheng-Kang
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-06-2013
• Subjects: Aqueous two-phase system; Archaeal Proteins - isolation & purification; Archaeal Proteins - metabolism; Bacteriorhodopsin; Bacteriorhodopsins - isolation & purification; Bacteriorhodopsins - metabolism; Cell Fractionation; Electrophoresis, Polyacrylamide Gel; Equipment Design; Halobacterium salinarum - chemistry; Halobacterium salinarum - metabolism; Phosphates - chemistry; Photochemistry - instrumentation; Photocurrent activity; Polyethylene Glycols - chemistry; Purple Membrane - chemistry; Purple Membrane - metabolism; Purple membrane purification; Ultracentrifugation; Bacteriorhodopsin; Purple membrane purification; Aqueous two-phase system; Photocurrent activity
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2013.03.011

•PM of halophilic bacteria could be completely collected at interface of PEG-phosphate two-phase system.•PM with high bacteriorhodopsin purity could be obtained.•Photocurrent activity of the PM was comparable to that obtained by sucrose gradient ultracentrifugation.
Purple membrane (PM) is a part of cytoplasmic membrane in certain extreme halophilic microorganisms belonging to Domain Archaea. It transduces light energy to generate proton gradient for ATP synthesis in the microorganisms. Bacteriorhodopsin (BR) is the only protein in PM responsible for the generation of proton gradient. Generally, PM was purified from Halobacterium salinarum via a tedious and lengthy sucrose density gradient ultracentrifugation (SGU). In this work, a facile method based on polyethyleneglycol (PEG)-phosphate aqueous-two- phase extraction system (ATPS) was employed to purify PM from cell lysate of H. salinarum. The results showed that PM could be completely recovered from the interface of PEG-phosphate ATPS with BR purity ca 94.1% as measured by UV-visible absorption spectra. In comparison with PM obtained by SGU, the PM isolated by ATPS could achieve the same level of purity and photocurrent activity (ca 177.2nA/μgBR/cm2) as analyzed by SDS–PAGE and photocurrent measurement, respectively. The easily scalable and straightforward ATPS procedure demonstrated that PM can be purified and recovered more cost-effectively with a significantly reduced operation time that should lead to broader range applications of PM possible.