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WO2002005922A1 - Procede pour extraire des proteines sous une forme pure a partir de plantes - Google Patents

Procede pour extraire des proteines sous une forme pure a partir de plantes Download PDF

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Publication number
WO2002005922A1
WO2002005922A1 PCT/DE2001/001997 DE0101997W WO0205922A1 WO 2002005922 A1 WO2002005922 A1 WO 2002005922A1 DE 0101997 W DE0101997 W DE 0101997W WO 0205922 A1 WO0205922 A1 WO 0205922A1
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WO
WIPO (PCT)
Prior art keywords
exchange chromatography
desired protein
protein
plant
buffer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/DE2001/001997
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German (de)
English (en)
Inventor
Dagmar Petsch
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
MPB Cologne GmbH Molecular Plant und Protein Biotechnology
Original Assignee
MPB Cologne GmbH Molecular Plant und Protein Biotechnology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by MPB Cologne GmbH Molecular Plant und Protein Biotechnology filed Critical MPB Cologne GmbH Molecular Plant und Protein Biotechnology
Priority to AU2001268942A priority Critical patent/AU2001268942A1/en
Publication of WO2002005922A1 publication Critical patent/WO2002005922A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/006—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from vegetable materials
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/009—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from unicellular algae
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/12—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from cereals, wheat, bran, or molasses
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/14—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds
    • A23J1/142—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds by extracting with organic solvents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/14—Vegetable proteins
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
    • B01D15/1807—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using counter-currents, e.g. fluidised beds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
    • B01D15/361—Ion-exchange
    • B01D15/362—Cation-exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
    • B01D15/361—Ion-exchange
    • B01D15/363—Anion-exchange
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • 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
    • C12N15/8258—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 for the production of oral vaccines (antigens) or immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient

Definitions

  • the present invention relates to a process for obtaining a desired native or recombinant protein from a plant in pure form, the desired protein being purified from the crude extract by means of expanded exchange technology using ion-exchange chromatography using expanded bed technology after obtaining a crude extract.
  • the method according to the invention is used for the isolation and purification of napin, napin-like 2S albumin, cruciferin, legumin-like IIS globulin or a recombinant scFv antibody.
  • Vegetable proteins are a hitherto rarely used reservoir of renewable raw materials. Vegetable storage proteins are obtained in particularly large quantities, for example in the production of oil from rapeseed, sunflowers and other oil plants, or from lupins or also in other agricultural processes. They are usually added to animal feed as a waste product, since no pure and thus technically usable components are available with technically and economically usable methods. Other vegetable proteins occur in smaller quantities, but their functionality is extremely interesting. So far, however, only a few vegetable proteins have been put to commercial use. This is due, among other things, to complex and therefore uneconomical cleaning processes which are also unsuitable for a larger scale. This contrasts with the extremely interesting properties, such as isolated vegetable storage proteins for various areas of application.
  • the two predominant storage proteins in rapeseed, napin and cruciferin which could previously only be obtained technically as a crude protein fraction, are suitable, for example, due to their physicochemical properties as isolated components for use as foams or adhesives (napin) or for film production (cruciferin). Furthermore, they are due to these properties also suitable for use in the food industry, e.g. as foaming agents and stabilizers.
  • the invention is therefore essentially based on the technical problem of providing a purification method for proteins from plants which has the disadvantages of the prior art does not have the process described in the art, ie above all it is simple, efficient and can be carried out on an industrial scale and also ensures that the desired proteins are of high purity and therefore preferably no further purification steps are required.
  • both native and recombinant proteins from plant tissue are separated into pure substances with high efficiency using the process according to the invention, which comprises an extraction step and a subsequent selective adsorption on cation and anion exchangers in the "expanded bed” technology or can be isolated as pure substances. It has been shown here that the degree of purity of the proteins is so high that an economically competitive production of new protein materials is also possible in comparison to plastic production based on fossil raw materials. It has also been shown that the otherwise necessary pre-treatment steps, such as degreasing of oil-containing seeds, or pre-precipitation steps are not necessary in the process according to the invention, but can, if appropriate, be combined therewith.
  • the process according to the invention can be carried out on an industrial scale. This has been shown by the separation of napin (albumin) and cruciferin (globulin) from rapeseed. Furthermore, it has been shown that the method according to the invention is also suitable for the use of foreign proteins, e.g. to isolate and purify scFv antibodies from transgenic plants. Reference is made to the examples below.
  • the present invention thus relates to a method for obtaining a desired native or recombinant protein from a plant in pure form, the method comprising the following steps: (a) disintegrating the plant to obtain a crude extract using a suitable extracting agent; and
  • the method according to the invention comprises only steps (a) and (b), i.e. there are no pretreatment and / or further purification steps, especially those that are based on specific physicochemical characteristics of the desired protein, e.g. Molecular weight, sedimentation coefficient, pl value, based, necessary.
  • pretreatment and / or cleaning steps can be added if necessary.
  • Suitable methods for digestion of the plant are known to the person skilled in the art and the latter can select suitable digestion methods according to the desired protein and the plant used. These can e.g. homogenization, such as in a grinder or mixer, and / or lysis with suitable lysis agents. Reference is made to the examples below.
  • the person skilled in the art also knows suitable extraction agents and selects them, inter alia, according to the known properties of the desired protein.
  • the extractant is preferably an aqueous solution, in particular phosphate buffer, TRIS buffer, MOPS buffer or an ethanolic extractant.
  • a denaturation and possibly renaturation step is optionally carried out before or after the extraction step if the desired protein should be in insoluble form.
  • Suitable denaturation processes and renaturation processes are known to the person skilled in the art and the person skilled in the art also knows the conditions which are required in these processes for the desired protein not to lose its biological activity or to regain it.
  • ion exchange chromatography is carried out using "expanded bed” technology in order to purify the desired protein.
  • "Expanded bed” technology is a very robust process that does not require any organic solvents or other high amounts of salt.
  • the "expanded bed” technology is easily transferable to the industrial scale and does not require any complex apparatus technology.
  • the adsorbents show no signs of "fouling", so that long service lives of the chromatography columns are guaranteed.
  • Straetkvern et al. Bioseparation 7 (1999), 333-345.
  • the "expanded bed” technology is not carried out in connection with affinity chromatography but, for the first time, with ion exchange chromatography.
  • ion exchange chromatography is carried out for the process according to the invention.
  • anion exchange or cation exchange chromatography is carried out for the process according to the invention.
  • the choice of the defined pH value for the binding of the protein to the chromatography material (loading) also depends on these properties.
  • the person skilled in the art selects the pH value on the basis of the pI value of the desired protein, the pI value either being calculated from the protein sequence or being determined by means of isoelectric focusing.
  • the pH value is chosen so that the desired protein has a surface charge opposite to the main contamination and the person skilled in the art will then select the sorbent suitable for the surface charge of the desired protein, ie a cation or anion exchanger.
  • the setting of a defined pH so that the protein components of a crude protein mixture contained in the extract can be selectively bound to the given chromatography material is of crucial importance. This makes it possible, for example, to purify two or more protein components, the pI values of which differ sufficiently, from a mixture.
  • the term "in pure form" as used herein means that the protein is essentially free of contaminants, preferably a purity of at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99 % having.
  • the method according to the invention is suitable for the purification of a desired protein from a plant of any plant species, i.e. it can be both a monocot and a dicot.
  • plant also includes gramineae, chenopodia, leguminous plants, Brasicaceae, Solanaceae, fungi, mosses and algae. They are preferably useful plants, e.g. plants such as wheat, barley, rice, corn, sugar beet, sugar cane, rapeseed, mustard, turnip, flax, pea, bean, lupine, tobacco and potato.
  • Any plant part or tissue of the plant can be used to obtain the protein, the selection being made according to the different concentration of the desired protein in the individual plant parts or tissues.
  • the desired protein is preferably obtained from seeds, leaves, tubers, root pieces, seedlings, cuttings, etc., with potato tubers being particularly preferred.
  • the desired protein is obtained from a storage protein mixture.
  • storage proteins which can be isolated and purified using the method according to the invention are napin, cruciferin, patatin, legumin and vicillin.
  • the binding of the desired protein to the ion exchange material takes place at a pH of 7.5 to 9, most preferably the desired protein is eluted with a buffer containing NaCl of defined molarity or by means of a NaCl gradients.
  • a buffer containing NaCl of defined molarity or by means of a NaCl gradients.
  • the A person skilled in the art can determine the optimal elution conditions on the basis of the known characteristics of the desired protein and / or by preliminary tests on a laboratory scale.
  • the person skilled in the art chooses (a) anion exchange chromatography or (b) cation exchange chromatography, for (a) preferably a chromatography material with the properties of Streamline DEAE TM or Streamline QXL (A ersham Pharmacia , Upsala, Sweden) and for (b) preferably Streamline SP XL TM (Amersham Pharmacia).
  • the method according to the invention allows, among other things, the isolation and purification of native proteins, for example napin, napin-like 2S albumin proteins, cruciferin and legumin-like IIS globulin proteins from plant seeds.
  • the napin-like proteins are proteins with an isoelectric point that is 8.0 or above, a molecular weight between 10 and 20 kDa and a composition of two heterodimeric subunits ( ⁇ and ⁇ chains), which are linked via one or more disulfide bridges.
  • Legumin-like IIS globulin proteins are characterized by an isoelectric point between 4.0 and 8.0, a molecular weight between 250 and 400 kD and a composition as hexamers from subunits, which are each composed of an ⁇ and ⁇ subunit, which in turn are linked by one or more disfuld bridges.
  • the desired proteins to be isolated are napin, napin-like 2S albumin proteins, cruciferin and legumin-like IIS globulins which, for example, from non-defatted rapeseed under the conditions specified in Example 1 below can be isolated and cleaned.
  • the proteins napin and cruciferin can be isolated and purified from the same crude extract, the surface charges of the proteins being adjusted by varying the pH so that only one of the two main components (napin and cruciferin) each aqueous rapeseed extract selectively interacts with anion and cation exchangers.
  • An efficient adsorption chromatographic separation can thus be achieved.
  • the method according to the invention also allows the isolation and purification of recombinant proteins, preferably scFv antibodies, which e.g. can be isolated and purified, as indicated in Examples 2 to 4 below, in which an aqueous extract of potato leaves was prepared, which was then introduced into an "expanded bed” process without precipitation or other pre-cleaning steps.
  • recombinant proteins preferably scFv antibodies
  • the method according to the invention is distinguished in many respects from conventional methods. Above all, its simplicity, technical scalability without high equipment expenditure, the robustness of the column materials, the possible elimination of oil removal from plant tissues such as seeds, and extensive pre-treatment steps before applying the aqueous extract to the chromatography material and the achievable high Purity of the native or recombinant proteins and thus their availability at economically favorable conditions. Furthermore, the order of isolation of several proteins from the same protein mixture can be chosen arbitrarily, taking into account the physicochemical characteristics of the respective proteins.
  • SDS page made from transgenic potato leaves A 10% Novex gel from Invitrogen (Groningen, NL) was used; Running buffer: MES, color: silver. Lane 1 shows the leaf extract, lane 2 the passage of the Streamline QXL column, lane 3 the eluate from the Streamline Q XL column, lane 4 is the wash fraction with 0.5 M NaCl. Lane 5 is the SDS-7 molecular weight marker from Sigma (Deisenhofen).
  • Rapeseed 100 g was ground in a grinder to medium grinding strength and extracted with five times the amount of buffer (20 mM phosphate buffer, pH 7.5). For this, the mixture was about 30 min. stirred and then the solid portions were centrifuged off. After decanting the supernatant into a collecting vessel, the precipitate was extracted a second time with the same amount of buffer as before and processed further as described above. The combined liquid phases were adjusted to a pH of 7.5 and then fed directly to the fluid bed adsorption ("expanded bed” technology). (B): Extraction of napin
  • charge buffer 20 mM phosphate buffer, pH 7.5
  • the cruciferin was obtained from the run of the napin adsorption from (B) by "expanded bed” adsorption.
  • the continuous fraction from (B) was adjusted to a pH of 8.5 and adsorbed on an anion exchanger (Streamline DEAE TM; Amersham Pharmacia).
  • Streamline DEAE TM Streamline DEAE TM; Amersham Pharmacia
  • the expanded state was then washed with 10 column volumes of charge buffer (20 mM phosphate buffer, pH 8.5). All other work steps were carried out in the sedimented state of the bed.
  • the purified storage proteins napin and cruciferin were subjected to a purity analysis using SDS-PAGE (see FIG. 1 for napin and FIG. 2 for cruciferin). In both cases the purity of the proteins is 99%.
  • Example 2 Isolation of recombinant scFv antibodies from transgenic potato leaves
  • Transgenic potato leaves transformed with a vector encoding an scFv antibody were mixed in a ratio of 1: 1 (mass: volume) with 20 mM phosphate buffer (pH 7.0) and homogenized in a "Warring Blender” for 3 6 seconds ,
  • Adsorption was carried out on Streamline Q XL TM (Amersham Pharmacia).
  • the expanded state was washed with 10 column volumes of loading buffer (20 mM phosphate buffer, pH 7.0) and 3 column volumes of loading buffer in the sedimented state of the bed, followed by elution of the scFv antibodies using a linear salt gradient from 0 to 0. 5 M NaCl in 20 mM phosphate buffer, pH 7.0.
  • the purified antibodies were desalted and lyophilized using methods known to those skilled in the art. SDS-PAGE was able to demonstrate that the main protein component of the leaves, ribulose bisphosphate carboxylase, was completely separated in one step and antibodies with a purity of 90% could be obtained (FIG. 3).
  • Example 3 Isolation of recombinant scFv antibodies from transgenic rapeseed
  • the expanded state was washed with 10 column volumes of loading buffer (20 mM phosphate buffer, pM 7.0) and 3 column volumes of loading buffer in the sedimented state of the bed, followed by elution of the scFv antibodies with 6 column volumes of washing buffer (phosphate buffer, pH 7, 5) with 0.5 M NaCl.
  • the highly purified scFv antibodies were desalted and lyophilized using methods known to those skilled in the art.
  • Example 4 Isolation of recombinant scFv antibodies from defatted transgenic rapeseed
  • Adsorption was carried out on Streamline Q XL TM (Amersham Pharmacia). After loading, the expanded state was washed with 10 column volumes of loading buffer (20 M phosphate buffer, pH 7.5) and 3 column volumes of loading buffer in the sedimented state of the bed, followed by elution of the scFv antibodies with 6 column volumes of loading buffer with 0.5 M NaCl.
  • the highly purified scFv antibodies were with the expert known processes desalted and lyophilized.

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Abstract

L'invention concerne un procédé pour extraire, sous une forme pure, une protéine souhaitée, native ou recombinée, à partir d'une plante. Après obtention d'un extrait brut par un moyen d'extraction approprié, la protéine souhaitée est retirée de l'extrait brut et purifiée par chromatographie par échange d'ions selon la technologie en lit expansé. La protéine souhaitée peut être par exemple la napine, l'albumine 2S de type napine, la cruciférine, la globuline 11S de type légumine ou un anticorps scFv recombiné.
PCT/DE2001/001997 2000-07-18 2001-05-23 Procede pour extraire des proteines sous une forme pure a partir de plantes Ceased WO2002005922A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001268942A AU2001268942A1 (en) 2000-07-18 2001-05-23 Method for extracting proteins from plants in pure form

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10035292.8 2000-07-18
DE2000135292 DE10035292A1 (de) 2000-07-18 2000-07-18 Verfahren zur Gewinnung von Proteinen aus Pflanzen in reiner Form

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WO2002005922A1 true WO2002005922A1 (fr) 2002-01-24

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005021764A3 (fr) * 2003-08-27 2005-05-19 Orf Liftaekni Ehf Processus de clivage proteolytique et purification de proteines de recombinaison
WO2008086811A1 (fr) * 2007-01-15 2008-07-24 Upfront Chromatography A/S Production de biocarburant et de protéine à partir d'une matière première
WO2009018660A1 (fr) * 2007-08-03 2009-02-12 Burcon Nutrascience (Mb) Corp. Production de la protéine de canola 2s par échange d'ions
DE102014005466A1 (de) 2014-04-12 2015-10-15 Klaus Düring Verfahren zur Gewinnung von Napin und Cruciferin oder einem Gemisch davon aus Raps
US10457704B2 (en) 2014-01-29 2019-10-29 Upfront Chromatography A/S Separation processes for pea protein
WO2020148704A1 (fr) 2019-01-18 2020-07-23 R. J. Reynolds Tobacco Company Purification de protéines rubisco dérivées de plantes

Families Citing this family (3)

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WO2005021762A3 (fr) * 2003-08-27 2005-05-19 Orf Liftaekni Ehf Processus de non denaturation pour la purification de proteines recombinantes provenant de vegetaux
US7462701B2 (en) 2003-08-27 2008-12-09 Orf Liftaekni Hf. Non-denaturing process to purify recombinant proteins from plants
US7834161B2 (en) 2003-08-27 2010-11-16 Orf Liftaekni Hf. Process for proteolytic cleavage and purification of recombinant proteins produced in plants
WO2005021764A3 (fr) * 2003-08-27 2005-05-19 Orf Liftaekni Ehf Processus de clivage proteolytique et purification de proteines de recombinaison
AU2008207207B2 (en) * 2007-01-15 2012-10-18 Upfront Chromatography A/S Production of biofuel and protein from a raw material
WO2008086811A1 (fr) * 2007-01-15 2008-07-24 Upfront Chromatography A/S Production de biocarburant et de protéine à partir d'une matière première
US8815551B2 (en) 2007-01-15 2014-08-26 Upfront Chromatography A/S Production of biofuel and protein from a raw material
JP2010515441A (ja) * 2007-01-15 2010-05-13 アップフロント・クロマトグラフィ・アクティーゼルスカブ 原材料からのバイオ燃料およびタンパク質の製造
RU2483111C2 (ru) * 2007-01-15 2013-05-27 Апфрант Кроматографи А/С Производство биотоплива и белка из сырья
AU2008286176B2 (en) * 2007-08-03 2013-02-07 Burcon Nutrascience (Mb) Corp. Production of 2S canola protein involving ion exchange
JP2010535207A (ja) * 2007-08-03 2010-11-18 バーコン ニュートラサイエンス （エムビー） コーポレイション イオン交換を含む、２ｓカノーラタンパク質の製造
US7750119B2 (en) 2007-08-03 2010-07-06 Burcon Nutrascience (Mb) Corp. Production of 2S canola protein involving ion exchange
CN101801999B (zh) * 2007-08-03 2013-10-30 伯康营养科学(Mb)公司 涉及离子交换的2s卡诺拉蛋白的生产
WO2009018660A1 (fr) * 2007-08-03 2009-02-12 Burcon Nutrascience (Mb) Corp. Production de la protéine de canola 2s par échange d'ions
US10457704B2 (en) 2014-01-29 2019-10-29 Upfront Chromatography A/S Separation processes for pea protein
DE102014005466A1 (de) 2014-04-12 2015-10-15 Klaus Düring Verfahren zur Gewinnung von Napin und Cruciferin oder einem Gemisch davon aus Raps
WO2015154884A1 (fr) 2014-04-12 2015-10-15 Pilot Pflanzenöltechnologie Magdeburg E.V. (Ppm E.V.) Procédé d'obtention de napine et de cruciférine ou d'un mélange de ces substances à partir de colza
CN106455623A (zh) * 2014-04-12 2017-02-22 皮洛特珀弗兰泽诺科技马格德堡公司（Ppm E.V.) 用于从菜籽获得napin和cruciferin或它们的混合物的方法
US10383345B2 (en) 2014-04-12 2019-08-20 Pilot Pflanzenöltechnologie Magdeburg e.V. Method for obtaining napin and cruciferin or a mixture thereof from rapeseed
WO2020148704A1 (fr) 2019-01-18 2020-07-23 R. J. Reynolds Tobacco Company Purification de protéines rubisco dérivées de plantes

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