WO2005121171A1 - Proteines stabilisant des molecules hydrophobes - Google Patents

Proteines stabilisant des molecules hydrophobes Download PDF

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Publication number
WO2005121171A1
WO2005121171A1 PCT/FI2005/000277 FI2005000277W WO2005121171A1 WO 2005121171 A1 WO2005121171 A1 WO 2005121171A1 FI 2005000277 W FI2005000277 W FI 2005000277W WO 2005121171 A1 WO2005121171 A1 WO 2005121171A1
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Prior art keywords
oleosin
oil
oil bodies
protection
nano
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PCT/FI2005/000277
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English (en)
Inventor
Petri Susi
Tony Wahlroos
Mauri Mäkelä
Fusao Tomita
Timo Korpela
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Petri Susi
Tony Wahlroos
Maekelae Mauri
Fusao Tomita
Timo Korpela
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Application filed by Petri Susi, Tony Wahlroos, Maekelae Mauri, Fusao Tomita, Timo Korpela filed Critical Petri Susi
Publication of WO2005121171A1 publication Critical patent/WO2005121171A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5052—Proteins, e.g. albumin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B5/00—Preserving by using additives, e.g. anti-oxidants
    • C11B5/0007—Organic substances
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B5/00—Preserving by using additives, e.g. anti-oxidants
    • C11B5/0021—Preserving by using additives, e.g. anti-oxidants containing oxygen
    • C11B5/0028—Carboxylic acids; Their derivates
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B5/00—Preserving by using additives, e.g. anti-oxidants
    • C11B5/0085—Substances of natural origin of unknown constitution, f.i. plant extracts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/86—Products or compounds obtained by genetic engineering

Definitions

  • the invention relates to methods enabling to prepare improved commodities by nanotechnology in the fields of food, feed, cosmetics, and medicine.
  • the invention relates to products, which benefit from improved stability of water-insoluble compounds like fats and oils or compounds dissolved in such hydrophobic materials.
  • the substances are protected by their encapsulation by proteins, which assemble to make a biphasic membrane layer between hydrophobic and hydrophilic phases.
  • lipids into plant seeds are essential not only for survival and subsequent growth and maturation of new plants but also to serve as an important food source for animals and humans. Seeds of many plant species store lipids in discrete organelles called oleosomes or oil bodies. Oil
  • TAG triacylglycerols
  • oleosins proteins and phospholipids.
  • Oil bodies are about 0.6-2 ⁇ m in diameter and contain a core of TAGs surrounded by an outer coat of phospholipid (PL) monolayer and a single class of proteins termed oleosins.
  • PL phospholipid
  • oleosins a single class of proteins termed oleosins.
  • Current evidence suggests that oleosin synthesis occurs via co-translational integration into endoplasmic reticulum (ER) membranes.
  • Biogenesis of oil bodies parallels the localisation of oleosins even though the signal sequence for targeting of oleosin to the sites where oil bodies are formed is not known. This indicates dynamic interaction between sites of oil body formation and oleosin synthesis as described (Sarmiento et al. 1997, Plant J 11:783-796).
  • Oleosin structure possesses three different functional domains. It is known in the prior art that the hydrophobic central domain is the key element for localisation of oleosin into oil bodies; lack of it totally abolishes localisation of oleosin into oil bodies (Abell BM, et all 997, Plant Cell
  • Hydrophobic central domain is flanked by the N- and C-terminal domains, which are variable in length (50-70 and 55-98 amino acids, respectively).
  • Short amino- (N)-terminal deletions (20 [N20] and 40 [N40] amino acids; respectively) do not disturb the transportation of oleosin into oil bodies, but longer N-terminal deletions (N66 and N90) reduce the localisation rate or fully abolish it.
  • N-terminal hydrophilic domains part are needed for targeting or localisation of oleosin into oil bodies.
  • hydrophobic central domain in intact oil bodies is predominantly ⁇ -helical in structure, and it can
  • oleosins complement the functions of some other lipid associated proteins (Hope et al. J. Biol. Chem. 277, 4261-4270).
  • the N- and C-terminal domains of oleosins have been predicted to contain amphipathic ⁇ -helix and some random-coil structure.
  • Oleosins accumulate with oil bodies, and oil seeds subjected to an aqueous extraction result in intact oil bodies (van Rooijen and Moloney, Bio/technol. 1995, 13:72-77). They can be separated with the accumulated oleosins by centrifugation from the rest of cellular debris as a floating oily "scum". Preparation of oleosomes and oleosome coats have been described (Bergfeld et al., 1978, Planta, 143:297-307). Mustard seeds were crushed with mortar and pestle followed by sucrose gradient centrifugation to get the oil body fraction.
  • Tzen and Huang J. Cell Biol. 1992, 117:327-335) showed that reconstitution of oil bodies was successful if oleosins, triacyl glycerol (TAG) and phospholipids (PL) were present. It was also reported that either TAG and PL purified from maize or commercially available lipids (dioleoyl phosphatidylcholine and a 1:2 molar mixture of triolein and trilinolein) could be used in the reconstitution.
  • TAG triacyl glycerol
  • PL phospholipids
  • WO 0226788 (Harada Takya et al.) describes the use of oleosin as a preferable safe natural stabilizer of emulsification to be applied with or without other emulsifying agents. Because of the generally known biphasic nature of oleosins, like with the detergents, the emulsifying function is evident. Normally emulsified or micellar fats and oils are more prone to chemical reactions, including oxidation, than their non-micellar counterparts due to the drastically higher surface area of the reacting species.
  • WO 0226788 differs essentially from that of the present invention, which states that oleosin can be used for protection of hydrophobic compounds from chemical reactions.
  • Provisional patent application JP2002-101820 describes the preparation of oil bodies from soya seeds by the centrifugation of water-soya suspensions by the methods essentially known from the prior art followed by a heat treatment. Said invention does not involve examples for the use of the prepared nanoparticles or does not describe their chemical or functional properties.
  • oleosin can stabilize oils and compounds dissolved in oils from chemical reactions. This finding was studied and found that oleosins form a tight three- dimensional network via intramolecular non-covalent linkages between oleosin molecules, which are partially embedded into lipids. This protein "coat” protects the lipid phase from the chemicals. It was especially surprising that the oleosin "coat” can protect lipids from molecular oxygen because molecular oxygen is a small molecule, which easily diffuses through pores of, for example, certain plastic films (PTFE and others) of even of high thicknesses. Thus, the oleosin coat forms a specific shield over lipid bodies. Comprehension of this oleosin coat structure enabled us to devise technology for protection of hydrophobic compounds by the oleosin, as well as developing new methods for producing oleosin.
  • FIG. 1 Stability of edible seed oil from B. rapa for oxidation caused by an intact or deactivated oil bodies.
  • the amount of conjugated dienes (measured by UV absorption at 234 nm in methanol) served as the measure of the oxidation (a).
  • the relative area of gas-solid chromatographic peaks of propanal indicates the volatile oxidation products generated by the reaction of unsaturated lipids with oxygen (b). Heat treated oleosin was used as the control.
  • oleosin-66-GFP terminal oleosin mutant fused to GFP
  • the inserted frame in the middle of the figure is magnified in c, d and e.
  • Figure 3 Molecular model of oleosins residing on the surface of oil bodies in water-lipid environment, (a) Longitudinal section of an oil body (sphere) covered by oleosin molecules, (b) Organization of oleosin molecules on the surface of oil bodies (a view from the top).
  • FIG. 1 Sequence of Arabidopsis thaliana oleosin gene (data bank ace. X62353).
  • the basic embodiment of the present invention is the finding that edible, technical or medical fats and oils, or other such hydrophobic compounds, can be protected from penetration of harmful substances by coating with specific proteins. Equally well, compounds dissolved or suspended in such hydrophobic materials can be protected.
  • the harmful substances can be chemically reactive or such which can spoil odour, taste, nutritive value, or other related properties.
  • a key methodological step leading to the present invention was the finding that a marker protein
  • Green Fluorescent Protein can be introduced into a specific position of the oleosin recombinant protein without losing functions of these two proteins.
  • N- and C-termini of two oleosins bind intermolecularly to each other to form a continuum of ⁇ -
  • oleosin-like proteins can be found in e.g. mammals and we assume that their mechanism of action is similar to zipper oleosins from plants. These proteins include adipophilin, perilipins, and caveolins.
  • viruses such as core proteins of Hepatitis C virus and GB virus-B contain related structures. These gene and amino acid sequences can be found from publicly available data banks and such data can be subjected to computer modeling procedures to find out the zipper structures.
  • Oleosin contains at least 40 percent of oil, which is extracted to produce margarines and oils. Oleosin can be directly extracted during the processes of seed oil production.
  • Oleosin as other lipid or membrane-associated proteins, has structural features that are difficult to determine with x-ray crystallography, and therefore other indirect non-trivial and novel techniques had to be applied to solve complex structural bias of oleosin. It is known from previous art that oleosin has structure of three different functional domains. Hydrophobic central domain is essential for localisation of oleosin into oil bodies, and it can even complement the function of an heterologous organism; binding of core protein of Hepatitis virus C to lipid membranes was restored by central domain of oleosin protein (Hope RG, et al. 2002, J Biol Chem 277:4261-4270).
  • Native oleosins can be extracted from seed materials by added oil droplets micellized into water with or without detergents. However, defatting of seeds by organic solvents tend to denaturate oleosin. Renaturation is a known technique in protein chemistry in transforming biologically inactive proteins and their aggregates back to biologically active forms. This technique is commonly used, for example, in the context of the proteins in the inclusion bodies produced by recombinant Escherichia coli. Usually, the renaturation of denaturated or aggregated proteins are unfolded by chaotropic salts, detergents, or other organic compounds in reducing conditions and then the unfolding agent is removed.
  • Oleosin is relatively small protein, which can refold whenever the conditions of the renaturation are met.
  • oleosin from defatted oil seeds can be extracted by oil bodies from aqueous solutions. Previously such renaturation of oleosin has not been considered possible.
  • refolding of oleosin can be created at the interfacial junction of lipid-water.
  • the basic embodiment of the present invention is the established and unique structure of oleosin in the lipid-water interphase, which enables to advise several useful functions and technologies for oleosins.
  • Oil bodies covered with truncated oleosin molecules were easily stained with neutral, lipid-specific stain, Nile Blue A, whereas oil bodies covered with intact oleosins were not.
  • Nile Blue A is a molecule with small molecular size of 730 Da (Spiekermann et al. 1999. Arch Microbiol 171 :73-80), also this result shows that surface of oil bodies is covered by a tight net of oleosin molecules that will not allow small molecules to pass or diffuse through. Thus, intact oleosin structure is required for protection of the lipid phase and preservation of structural integrity of oil bodies.
  • central hydrophobic domain has been shown to be an ⁇ -helix with FT-IR or CD
  • residues in these ⁇ -helices interact with phospholipids on the surface of oil bodies, and the negatively charged residues are oriented towards the exterior.
  • sequence of residues 162-167 which is located after two amphipathic ⁇ -helical structures, is very similar to sequence of residues 23-28 of N-terminal domain. Both of these segments are extremely rare among known proteins; besides known oleosin structures, homological sequences were identified only against segment 162-167 in synaptotagmin C, which is a synaptic vesicle protein and a putative trigger of exocytosis in animal systems (Zhang et al. 2002, Genesis 34:142-145). This implies indirectly that domains found in oleosin protein are important for protein function particularly in water- lipid environment.
  • segment of residues 162-167 of oleosin protein does not possess ⁇ -helical or ⁇ -strand structures. However, because of the distribution of the
  • the central hydrophobic domain of oleosin may be essential in binding the zipper assembly onto lipid bodies, it is evident that the central part of oleosin can be readily replaced with another hydrophobic domain by the methods of gene engineering. Then the protein is to be considered different from any known oleosin and functionally different from recombinant oleosins described in the prior art.
  • the zipper domains themselves, without the central domain have a weak affinity to the lipid interphase which can be adequate for protection from certain chemical reactions.
  • the recombinant zipper proteins involving the central domain of desired properties can be produced by the known gene-engineering techniques.
  • the found zipper structure can have a variability of applications in biotechnology other than the protection of lipids from chemical reactions of oxygen and other chemicals. Not only lipid interfaces can be covered by these structures but also any hydrophobic - hydrophilic interface. In such cases the anchoring centre domain can be artificially designed by computer modelling.
  • the counterparts of the zipper can be in the same molecule like in oleosin or both of them can carry different anchoring domains.
  • the zipper cover can be used for prevention of molecular trafficking from or into hydrophobic phase. Usually such artificial covers are produced by gene-
  • oleosin-GFP fusion Full-length oleosin-GFP fusion was described earlier (Wahlroos et al. 2003, Genesis 35:125- 132). The oleosin sequence is shown in the figure 4. Arabidopsis thaliana oleosin gene carrying N-terminal deletion of 66 amino acids was created with PCR and cloned in-frame with GFP. Primers for PCR of oleosins were: OLE66-forw: 5'-GCACCATGGTTGGAACTGTCATA OLE66-rev: 5 '-GAACTCGAGAAGTAGTGTTGCTG.
  • Tobacco plants were grown in soil in a greenhouse at 24 °C. Plants were illuminated using fluorescent lights (Lucalox®, type LU400/HO/T/40) operated on a 16 h day photoperiod. Mature, full-expanded, leaves were subjected to particle bombardment. Particle bombardment was performed by the rupture disk method with a high-pressure helium-based apparatus PDS-1000 (Bio-Rad). Tungsten particles were prepared by vortexing of 60 mg in 70 % ethanol for 3-5 minutes, followed by incubation on a bench for 15 min. Mixture was pelleted by short spinning and the supernatant was removed. Sterile water was added onto pellet and vortexed for 1 min.
  • Particles were allowed to settle for 1 minute and spirmed for 2 s. The supernatant was removed. This was repeated three times. Sterile 50 % glycerol was added to bring particle concentration to 60 mg/ml. For each particle bombardment, DNA was precipitated
  • DNA 50 ⁇ l of CaCl 2 (2.5 M) and 20 ⁇ l of spermidine (0.1 M) were mixed and vortexed for 2-3
  • a detached leaf of Nicotiana benthamiana (15-30 mm size) was placed in the center of a plastic Petri dish and bombarded on a solid support at a target distance of 7 cm. Bombardment was done with a pulse of 1350 kPa helium gas in a vacuum chamber. Inoculated leaves were analyzed 24 hours after particle bombardment. GFP fluorescence was monitored using a confocal laser scanning imaging system MRC-1024 (Bio-Rad).
  • Tobacco leaf cells expressing oleosin-66-GFP were infiltrated with an aqueous solution (lOOng ml '1 ) of fluorescing, lipid-specific stain, Nile blue A (Sigma-Aldrich, Helsinki, Finland; Spiekerman et al. 1999, Arch Microbiol 171:73-80), 24 h after particle bombardment.
  • GFP fluorescence and Nile blue A fluorescence were visualised using appropriate confocal microscope fluorescence filters as done earlier (Wahlroos et al. 2003, Genesis 35:125-132).
  • Oxidation stability tests were done with intact and heat deactivated product. Heat-deactivation was done at 90 °C for 5 min under nitrogen atmosphere. Stability test was done in closed test tubes at 37 °C. Oxidation was followed three times per week analysing conjugated dienes and volatile oxidation products, such as propanal, which are typical lipid oxidation products (Chan and Coxon 1987, Academic Press Inc., London, pp 17-50). Conjugated dienes were measured spectrophotometrically at 234 nm in methanol. Volatile compounds were measured with headspace gas clrromatograph (Autosystem XL, Perkin Elmer, USA). Each measurement was repeated twice ( Figure la and lb).
  • oil seeds was used for the preparation of oil bodies, including soya, rape, false flax (Camelina sativa), oats, canola (Brassica napus), spring turnip rape (Brassica rapa), and sesam oil.
  • the seeds were used directly or after cold processing removing the majority of the oil while leaving plant remnants and proteins in the pellet. Oleosin in defatted (by extraction with organic solvents) pellet could be regenerated by extraction with oil micelles.
  • the seeds are crushed and made mechanically into a fine powder of less than 50-100 ⁇ m diameter on the average (controlled by microscopy).
  • the powder (1 kg) is dispensed into water (10 liter) by strong mechanical agitation, which optionally can also involve ultrasonic oscillator treatment at a high energy. Temperature of the solution must stay below 60°C during the process.
  • the suspension is conditioned for 1- 5 h before and allowed to cool to +0-5°C.
  • the suspension is then filtrated using a standard drum filtration equipment operating at a reduced pressure.
  • the optimal filter is of about the similar pore size as is used for the recovery of bakery yeast.
  • the filter can be covered with diatomaceous earth for preventing clogging of the filter.
  • the substance recovered from the blade of the drum filter can be returned to the process after homogenization and refiltrated to increase the yield of oil bodies/seed mass.
  • the filtrate contains remnants of oil bodies and soluble seed proteins including oleosin.
  • the oil body fraction is not necessary to purify the oil body fraction as described in the technologies described in the context of known centrifugation methods of preparing oil bodies.
  • the oil bodies can be stored for several months at temperatures near 0°C.
  • the water content of the oil bodies is kept at 10-15 %.
  • the ratio of oil/hydrophobic material per protein depends on the application of the preparation. In many applications it is not necessary to purify the protein fraction. However, if it is needed the content and purity of oleosin can be controlled by the temperature, strength, and time of treatment of the first extraction process.
  • the minimum content of protein in oil bodies is 5 % (w/w, protein/ lipid).
  • TMV-MP is a known ⁇ -helical, hydrophobic membrane protein which binds to cellular membranes (Brill et al. 2000, Proc Natl Acad Sci USA 97, 7112-7117).
  • the N30K and C30K primers contained an EcoRI restriction site and Xhol restriction site, respectively.
  • the first strand cDNA was synthesized from TMV genomic RNA using 20 nM of C30K primer, 1 mM dNTPs and 1 unit of AMV in IX AMV reverse transcriptase (RT) reaction buffer (50 mM Tris-HCl; 8.3, 50 mM KC1, 10 mM MgCl 2 , 0.5 mM spermidine, 10 mM DTT) for TMV genomic RNA using 20 nM of C30K primer, 1 mM dNTPs and 1 unit of AMV in IX AMV reverse transcriptase (RT) reaction buffer (50 mM Tris-HCl; 8.3, 50 mM KC1, 10 mM MgCl 2 , 0.5 mM spermidine, 10 mM DTT) for TMV genomic RNA using 20 nM of C30K primer, 1 mM dNTPs
  • the template was denatured with heating for 3 min at 95°C, and 28 cycles of PCR were carried out with iCycler (Bio-Rad) thermal cycler with denaturation at 95°C for 1 min, primer annealing at 68°C for 1.5 min, primer extension at 72°C for 2 min, with a final elongation step after 30 cycles at 72°C for 10 min.
  • the resulting DNA product was cleaved by EcoRI and Xhol and cloned into EcoRI-JttoI-digested pG ⁇ M-7Zf(+) (Promega Corporation, USA; catalogue number P2251).
  • plasmid pGEM-30K was selected for further work.
  • the termination codon of the TMV 3 OK gene was replaced by a Xliol site for subsequent fusion with N- and C- termini of oleosin.
  • Oleosion-specific primers were designed according to full-length oleosin (Fig. 4.). This Constructs N-MP-C-ole was used to constructs artificial oil body membranes.
  • Vitamin A dissolved in olive oil (50 mg/ml) in the presence of lecitin (1209 mg/ml of olive oil) is micellized in ratio 1:20 (v/v) to water with a stirrer (20,000 rpm) on ice in nitrogen atmosphere.
  • Two grams of oil bodies (Example 8) is added to the mixture in the same conditions.
  • a control is made equally without oil bodies.
  • the liquids were allowed to stand at the room temperature and were analyzed by UV at the absorption maximum of vitamin A.
  • the liquid containing oil bodies were more stable for oxidation of vitamin A than the control.

Abstract

L'invention concerne des nanofilms protEiques spEcifiques sur des interfaces hydrophile-hydrophobe. On a trouvE que des protEines spEcifiques peuvent former des couches minces molEculaires sur des surfaces hydrophobes incurvEes ou planes qui empEchent efficacement un transfert de matiEre au travers des nano films. L'invention peut Etre utilisEe pour protEger contre l'oxygEne gazeux ou dissous des corps gras et des huiles, de mEme que des composEs dissous ou en suspension dans ceux-ci.
PCT/FI2005/000277 2004-06-14 2005-06-14 Proteines stabilisant des molecules hydrophobes WO2005121171A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2672953A1 (fr) * 2011-02-07 2013-12-18 Commonwealth Scientific & Industrial Research Organisation ( C.S.I.R.O. ) Corps huileux artificiels
WO2022021704A1 (fr) * 2020-07-30 2022-02-03 齐鲁工业大学 Membrane monocouche polypeptidique hydrophobe à potentiel élevé, procédé de préparation et application associés

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DATABASE WPI Week 200252, Derwent World Patents Index; AN 2002-483256 *
LEFEVRE T. AND SUBIRADE M.: "Formation of intermolecular beta-sheet structures: a phenomenon relevant to protein film structure at oil-water interfaces of emulsions", JOURNAL OF COLLOID AND INTERFACE SCIENCE, vol. 263, no. 1, July 2003 (2003-07-01), pages 59 - 67, XP027432822, DOI: doi:10.1016/S0021-9797(03)00252-2 *
LI M. ET AL: "Purification and structural characterization of the central hydrophobic domain of oleosin", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 277, no. 40, October 2002 (2002-10-01), pages 37888 - 37895 *
WANG L. AND TAO B.: "Formation and Properties of Soy Oil Body Oleosin-Based Edible Emulsion Films", 95TH AOCS ANNUAL MEETING & EXPO, 9 May 2004 (2004-05-09) - 12 May 2004 (2004-05-12), CINCINNATI, OHIO, USA, Retrieved from the Internet <URL:http://www.aocs.org/archives/am2004/session.asp?session=PCP+5%3A+General+Proteins+and+Co%2DProducts> [retrieved on 20050908] *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2672953A1 (fr) * 2011-02-07 2013-12-18 Commonwealth Scientific & Industrial Research Organisation ( C.S.I.R.O. ) Corps huileux artificiels
EP2672953A4 (fr) * 2011-02-07 2015-12-02 Commw Scient Ind Res Org Corps huileux artificiels
WO2022021704A1 (fr) * 2020-07-30 2022-02-03 齐鲁工业大学 Membrane monocouche polypeptidique hydrophobe à potentiel élevé, procédé de préparation et application associés

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