A unique caleosin serving as the major integral protein in oil bodies isolated from Chlorella sp. cells cultured with limited nitrogen.

Accumulation of oil bodies was successfully induced in a microalga, Chlorella sp., cultured in a nitrogen-limited medium. The oil bodies were initially assembled as many small entities (mostly 0.1-1 μm), and lately found as a major irregular compartment (>3 μm) occupying more than half of the cell space. Approximately, two thirds of oil bodies isolated from Chlorella cells were broken and formed a transparent oil layer on top of the milky compact layer of the remaining stable oil bodies after being washed with 0.1% triton X-100. The stable oil bodies mainly comprised triacylglycerols as examined by thin layer chromatography analysis and confirmed by both Nile red and BODIPY stainings. Integrity of these stable oil bodies was maintained via electronegative repulsion and steric hindrance possibly provided by their surface proteins. Immunological cross-recognition revealed that a major protein of 29 kDa, tentatively identified as caleosin, was exclusively present in Chlorella oil bodies. Mass spectrometric analysis showed that the putative caleosin possessed a trypic fragment of 13 residues matching to that of a hypothetical caleosin in Picea sitchensis. With the aid of a degenerate primer designed according to the tryptic peptide, a complete cDNA fragment encoding this putative caleosin was obtained by PCR. Phylogenetic tree analysis supports that Chlorella caleosin is the most primitive caleosin found in oil bodies to date.