Purification and Biophysical Characterization of the Photosystem I Complex from Botryococcus braunii

Abstract

Botryococcus braunii (B. braunii) is a green photosynthetic microalga that is highly investigated due to its capacity of hydrocarbon accumulation, secretion, and synthesis. However, its photosynthetic apparatus has not been elucidated. Photosystem I (PSI) is a membrane‐bound pigment‐protein complex that mediates the electron transfer from plastocyanin on the lumenal side to ferredoxin on the cytoplasmic side. In this study, our purpose was to isolate, purify and characterize PSI core subunits and light harvesting proteins. After the initial isolation via differential ultracentrifugation and solubilization of thylakoid membranes with 1% of n‐dodecyl β‐D‐maltoside, the resulting supernatant was loaded onto a Toyopearl DEAE‐650 ion exchange chromatography column. The purified PSI fraction obtained was pooled and concentrated using an Amicon centrifugal filter unit. The resulting protein concentration was 1 mg/ml, and the PSI characteristic 677 nm absorption maximum was attained. The biophysical characterization was performed via UV/Vis and fluorescence spectroscopy, native and SDS‐polyacrylamide gel electrophoresis, and MALDI‐MS, with the characteristic peaks and molecular weight results of the proteins and subunits found in PSI. Our characterization results show that PSI has been successfully purified, which may provide the fundamental knowledge of the function and possible applications of this integral membrane protein complex in B. braunii.Support or Funding InformationThis research was funded by NIH Research Initiative for Scientific Enhancement (RISE) Program Grant No. 5R25GM061151‐14 and DOD Contract No. W911NF‐11‐1‐0218.