Liquid-Liquid Phase Separation of Oil Bodies from Seeds
Fundamentally, oil bodies are discrete storage organelles found in oilseeds, comprising a hydrophobic triacylglycerol core surrounded by a half-unit phospholipid membrane and an outer shell of specialized proteins known as oleosins. Oil bodies possess a number of attributes that were exploited by SemBioSys Genetics to isolate highly enriched fractions of oil bodies through liquid-liquid phase separation for a number of commercial applications. The current chapter provides a general guide for the isolation of oil bodies from Arabidopsis and/or safflower seed, from which protocols can be refined for different oilseed sources. For SemBioSys Genetic’s recombinant technology, therapeutic proteins were covalently attached to oleosins or fused in-frame with ligands which bound oil bodies, facilitating their recovery to high levels of purity during “upstream processing” of transformed seed. Core to this technology was oil body isolation consisting of simple manipulation including homogenization of seeds to free the oil bodies, followed by the removal of insoluble fractions, and phase separation to recover the oil bodies. During oil body enrichment (an increase in oil body content concomitant with removal of impurities), a number of options and tips are provided to aid researchers in the manipulation and monitoring of these robust organelles.
KeywordsOil body Liquid phase separation Oilseeds Recombinant oil body Targeted oil body
- 11.Yurchenko OP, Field CJ, Nykiforuk CL et al (2007) Genetic modification of oilseeds to produce bioactive lipids. In: Acharya SN, Thomas JE (eds) Advances in medicinal plant research. Research Signpost, Trivandrum, pp 357–399Google Scholar
- 21.Fischer JJ, Nykiforuk CL, Chen X et al (2014) Delayed oxidation of polyunsaturated fatty acids encapsulated in safflower (Carthamus tinctorius) oil bodies. IJESIT 3(3):512–522Google Scholar
- 22.Markley N, Nykiforuk C, Boothe J et al (2006) Producing proteins using transgenic oilbody-oleosin technology. BioPharm Int 19(6):34–47Google Scholar
- 27.Nykiforuk CL, Shen Y, Murray E et al (2012) Plant-derived manufacturing of Apolipoprotein AI Milano: purification and functional characterization. In: Bertolini J, Goss N, Curling J (eds) Production of plasma proteins for therapeutic use. pp 283–300Google Scholar
- 32.Deckers HM, van Rooijen F, Boothe J et al (2000) Uses of oil bodies. US patent 6,146,645Google Scholar