Liquid-Liquid Phase Separation of Oil Bodies from Seeds

  • Cory L. NykiforukEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1385)


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.


Oil body Liquid phase separation Oilseeds Recombinant oil body Targeted oil body 


  1. 1.
    Chapman KD, Dyer JM, Mullen RT (2012) Biogenesis and functions of lipid droplets in plants: thematic review series: lipid droplet synthesis and metabolism: from Yeast to Man. J Lipid Res 53(2):215–226CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Huang AHC (1992) Oil bodies and oleosins in seeds. Annu Rev Plant Physiol Plant Mol Biol 43:177–200CrossRefGoogle Scholar
  3. 3.
    Tzen JTC (2012) Integral proteins in plant oil bodies. ISRN Botany. doi: 10.5402/2012/173954 Google Scholar
  4. 4.
    Vindigni J-D, Wien F, Giuliani A et al (2013) Fold of an oleosin targeted to cellular oil bodies. Biochim Biophys Acta 1828:1881–1888CrossRefPubMedGoogle Scholar
  5. 5.
    Hsieh K, Huang AH (2004) Endoplasmic reticulum, oleosins, and oils in seeds and tapetum cells. Plant Physiol 136:3427–3434CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Sarmiento C, Ross JH, Herman E et al (1997) Expression and subcellular targeting of a soybean oleosin in transgenic rapeseed. Implications for the mechanism of oil-body formation in seeds. Plant J 11:783–796CrossRefPubMedGoogle Scholar
  7. 7.
    Tzen JTC, Huang AHC (1992) Characterization of the charged components and their topology on the surface of plant seed oil bodies. J Biol Chem 267(22):15626–15634PubMedGoogle Scholar
  8. 8.
    Wu YY, Chou YR, Wang CS et al (2010) Different effects on triacylglycerol packaging to oil bodies in transgenic rice seeds by specifically eliminating one of their two oleosin isoforms. Plant Physiol Biochem 48:81–89CrossRefPubMedGoogle Scholar
  9. 9.
    Siloto RMP, Findlay K, Lopez-Villalobos A et al (2006) The accumulation of oleosins determines the size of seed oilbodies in Arabidopsis. Plant Cell 18:1961–1974CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Shimada TL, Chimada T, Takahashi H et al (2008) A novel role for oleosins in freezing tolerance of oilseeds in Arabidopsis thaliana. Plant J 55:798–809CrossRefPubMedGoogle Scholar
  11. 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
  12. 12.
    Carlsson AS, Zhu L-H, Andersson M et al (2014) Platform crops amenable to genetic engineering—a requirement for successful production of bio-industrial oils through genetic engineering. Biocatal Agric Biotechnol 3(1):58. doi: 10.1016/j.bcab.2013.007 Google Scholar
  13. 13.
    Damude HG, Kinney AJ (2008) Engineering oilseeds to produce nutritional fatty acids. Physiol Plant 132:1–10PubMedGoogle Scholar
  14. 14.
    Dyer JM, Mullen RT (2008) Engineering plant oils as high-value industrial feedstocks for biorefining: the need for underpinning cell biology research. Physiol Plant 132:11–22PubMedGoogle Scholar
  15. 15.
    Haslam RP, Ruiz-Lopez N, Eastmond P et al (2012) The modification of plant oil composition via metabolic engineering—better nutrition by design. Plant Biotechnol J. doi: 10.1111/pbi.12012 Google Scholar
  16. 16.
    Lopez NR, Haslam RP, Usher SL et al (2013) Reconstitution of EPA and DHA biosynthesis in Arabidopsis: iterative metabolic engineering for the synthesis of n-3 LC-PUFAs in transgenic plants. Metab Eng 17:30–41. doi: 10.1016/j.ymben.2013.03.001 CrossRefGoogle Scholar
  17. 17.
    Maheshwari P, Kovalchuk I (2014) Genetic engineering of oilseed crops. Biocatal Agric Biotechnol 3:31–37. doi: 10.1016/j.bcab.2013.11.001 Google Scholar
  18. 18.
    Nykiforuk CL, Shewmaker C, Harry I et al (2012) High level accumulation of gamma linolenic acid (C13:3Δ6,9,12 cis) in transgenic safflower (Carthamus tinctorius) seeds. Transgenic Res 21:367–381CrossRefPubMedGoogle Scholar
  19. 19.
    Nykiforuk CL, Furukawa-Stoffer TL, Huff PW et al (2002) Characterization of cDNAs encoding diacylglycerol acyltransferase from cultures of Brassica napus and sucrose-mediated induction of enzyme biosynthesis. Biochim Biophys Acta 1580(2-3):95–109CrossRefPubMedGoogle Scholar
  20. 20.
    Fisk ID, White DA, Lad M et al (2008) Oxidative stability of sunflower oil bodies. Eur J Lipid Sci Technol 110:962–968CrossRefGoogle Scholar
  21. 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. 22.
    Markley N, Nykiforuk C, Boothe J et al (2006) Producing proteins using transgenic oilbody-oleosin technology. BioPharm Int 19(6):34–47Google Scholar
  23. 23.
    Nykiforuk CL, Boothe JG (2012) Transgenic expression of therapeutic proteins in Arabidopsis thaliana seed. In: Voynov V, Caravella JA (eds) Therapeutic proteins: methods and protocols. Methods in molecular biology, vol 899. Springer. New York doi:  10.1007/978-1-61779-921-1_16 Google Scholar
  24. 24.
    Boothe J, Nykiforuk C, Shen Y et al (2010) Seed-based expression systems for plant molecular farming. Plant Biotechnol J 8:588–606CrossRefPubMedGoogle Scholar
  25. 25.
    Nykiforuk CL, Boothe JG, Murray EW et al (2006) Transgenic expression and recovery of biologically active recombinant human insulin from Arabidopsis thaliana seeds. Plant Biotechnol J 4:77–85CrossRefPubMedGoogle Scholar
  26. 26.
    Nykiforuk CL, Shen Y, Murray EW et al (2011) Expression and recovery of biologically active recombinant Apolipoprotein AIMilano from transgenic safflower (Carthamus tinctorius) seeds. Plant Biotechnol J 9:250–263CrossRefPubMedGoogle Scholar
  27. 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
  28. 28.
    Austin JR, Frost E, Vidi P-A et al (2006) Plastoglobules are lipoprotein subcompartments of the chloroplast that are permanently coupled to thylakoid membranes and contain biosynthetic enzymes. Plant Cell 18:1693–1703CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Shanmugabalaji V, Besagni C, Piller LE et al (2013) Dual targeting of a mature plastoglobulin/fibrillin fusion protein to chloroplast plastoglobules and thylakoids in transplastomic tobacco plants. Plant Mol Biol 81(1-2):13–25CrossRefPubMedGoogle Scholar
  30. 30.
    Vidi P-A, Kessler F, Bréhélin C (2007) Plastoglobules: a new address for targeting recombinant proteins in the chloroplast. BMC Biotechnol. doi: 10.1186/1472-6750-7-4 PubMedPubMedCentralGoogle Scholar
  31. 31.
    Han Z, Madzak C, Su WW (2012) Tunable Nano-oleosomes derived from engineered Yarrowia lipolytica. Biotechnol Bioeng 110:702–710CrossRefPubMedGoogle Scholar
  32. 32.
    Deckers HM, van Rooijen F, Boothe J et al (2000) Uses of oil bodies. US patent 6,146,645Google Scholar
  33. 33.
    Huang AH (1996) Oleosins and oil bodies in seeds and other organs. Plant Physiol 110:1055–1061CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Zienkiewicz K, Castro AJ, Alche JD et al (2010) Identification and localization of a caleosin in olive (olea europaea L.) pollen during in vitro germination. J Exp Bot 61:1537–1546CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Liu WX, Liu HL, Qu LQ (2013) Embryo-specific expression of soybean oleosin altered oil body morphogenesis and increased lipid content in transgenic rice seeds. Theor Appl Genet 126:2289–2297CrossRefPubMedGoogle Scholar
  36. 36.
    Nykiforuk CL, Johnson-Flanagan AM (1999) Storage reserve mobilization during low temperature germination and early seedling growth in Brassica napus. Plant Physiol Biochem 37(12):939–947CrossRefGoogle Scholar
  37. 37.
    Tzen JTC, Peng CC, Cheng DJ et al (1997) A new method for seed oil body purification and examination of oil body integrity following germination. J Biochem 121(4):762–768CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.SemBioSys Genetics Inc.CalgaryCanada

Personalised recommendations