WO1995001989A1 - Separation of proteins - Google Patents
Separation of proteins Download PDFInfo
- Publication number
- WO1995001989A1 WO1995001989A1 PCT/DK1994/000256 DK9400256W WO9501989A1 WO 1995001989 A1 WO1995001989 A1 WO 1995001989A1 DK 9400256 W DK9400256 W DK 9400256W WO 9501989 A1 WO9501989 A1 WO 9501989A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- protein
- peg
- crystallization
- added
- concentration
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
- C07K1/306—Extraction; Separation; Purification by precipitation by crystallization
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/814—Enzyme separation or purification
Definitions
- the present invention relates to a method of separating a protein, in particular an enzyme, from an aqueous solution of proteins, and recovery of the desired protein on crystalline form.
- Enzymes are usually provided as liquids or amorphous materials for industrial purposes. When not provided as liquids, they are usually provided as amorphous materials, because the known methods for crystallization of enzymes are usually regarded as too expensive to be used in an industrial scale. Due to the high purity of enzyme crystals, the provision of a cheap and simple method for crystallization of enzymes which is easily adaptable to an industrial scale is clearly a desideratum in the industry.
- Characteristic features of the hitherto known protein crystallization processes are very pure and con- centrated initial solutions, low yield, very long crystalliza ⁇ tion time, high consumption of chemicals including organic solvents, and poor industrial adaptability.
- the present invention provides a method for separating a protein from an aqueous mixture of proteins comprising
- Fig. 1 shows the peroxidase crystallization yield (%) at various PEG 4000 concentrations and various CaCl 2 concentrations ( ⁇ 1% CaCl 2 ; A 2% CaCl 2 and ° 2.5% CaCl 2 ) .
- Fig. 2 shows the peroxidase crystallization yield (%) at various pH values.
- Fig. 3 shows the peroxidase crystallization yield (%) at various enzyme and PEG 4000 concentrations ( ⁇ 3% peroxidase and 1.4% other enzymes; ⁇ 4.8% peroxidase and 2.3% other enzymes and A 6.7% peroxidase and 3.1% other enzymes) .
- Fig. 4 shows the catalase crystallization yield (%) at various CaCl 2 - concentrations and at various PEG 4000 concentrations ( ⁇ 20% PEG 4000; ⁇ 22% PEG 4000; A 24% PEG 4000; ° 26% PEG 4000; 0 28% PEG 4000 and ⁇ 30% PEG 4000) .
- Fig. 5 shows the catalase crystallization yield (%) at various CaCl 2 - concentrations and at various PEG 4000 concentrations ( ⁇ 20% PEG 4000 and ⁇ 25% PEG 4000) .
- Fig. 6 shows the catalase crystallization yield at various PEG 1500 concentrations and a CaCl 2 - concentration of 0.2 %.
- Fig. 7 shows the protease crystallization yield (%) at various PEG 4000 - concentrations.
- Fig. 8 shows the protease crystallization yield (%) at various PEG 4000 - concentrations and with a CaCl 2 - concentration of 1.1%.
- the present invention provides a method for separat ⁇ ing a protein or a polypeptide from an aqueous mixture compris ⁇ ing other proteins with different crystallization properties. It has surprisingly been found that by adding a water soluble polymer, e.g. polyethylene glycol, a protein may become separated from its mixture with other proteins and other impurities such as carbohydrate compounds, and precipitate on crystalline form.
- a water soluble polymer e.g. polyethylene glycol
- PEG polyethylene glycol
- PEG protein crystallization
- Another use of PEG for protein crystallization has been the creation of an aqueous 2-phase system by the use of PEG and high salt concentrations, in which the protein is concentrated in the PEG phase, nucleation takes place in the interphase and the crystal growth is occurring in the water phase with the driving force being the high salt concentration, for reference see Eur. J. Biochem. 189, 1990, pp. 1-23.
- the crystals then apparently take form in the droplets until they reach a size where they might burst out of the droplets generating a normal one phase aqueous system with solid crystals at the end of the crystallization.
- the system can as well end up as a 2-phase system with the bulk part of the crystals still in the droplet phase. Examples of crystal growth occurring both in the droplet phase and from the surface of the droplets out in the surrounding solution have also been observed.
- the method of the invention can be applied to separation of a protein from a mixture of proteins, in par ⁇ ticular to separation of an enzyme from a mixture of proteins.
- the enzyme containing solution is a culture broth obtained by cultivation of an enzyme producing microorganism.
- the method of the invention is applied to a culture broth that has first been subjected to solid/liquid separatory techniques, e.g. by flocculation, centrifugation, filtration or micro filtration. It may also be useful to purify by precipitation or use of chromatographic methods before crystallization.
- the method of invention comprises concentration of the enzyme containing solution by methods known per se. Such methods include con ⁇ centration by ultrafiltration, by diafiltration, by dialysa- tion, or by evaporation.
- the enzyme containing solution may be concentrated to a content of enzyme protein of from 0.1 to 25% w/w, more preferred of from 0.5 to 15% w/w, most preferred of from 1 to 10% w/w.
- the method is applied to separation of oxidoreductases, proteases, lipases, amylases and cellulases.
- Oxidoreductases which are defined and described in "Enzyme Nomenclature 1992, Academic press, Inc., San Diego", belong to a class of enzymes that catalyses transfer of electrons from one substance to another (oxidation-reduction) .
- Oxidoreductases include dehydrogenases, reductases, oxidases, transhydrogenases, catalases, peroxidases, and oxygenases. More specific examples include horseradish per ⁇ oxidase, ligninases and other peroxidases, and oxidases such as laccases . These enzymes are preferably of microbial origin.
- microorganism genera which may be used 5 for production of suitable oxidoreductases are: Trametes, Rhizoctonia, Pseudomonas, Bacillus, Streptomyces, Hy ⁇ rophorus, Coprinus, Polyporus, Candida, Curvularia, Cercospora, Myco- liophtora, Aspergillus, and Scvtalidium.
- the invention is not restricted to enzymes derived from the above 10 mentioned taxa. All microorganisms producing oxidoreductases with the desired properties may be used in relation to this invention.
- the oxidoreductase is preferably a laccase (EC 1.10.3.2) , a catalase (EC 1.11.1.6) , a peroxidase (EC 151.11.1.7) , or an oxidase.
- the method of invention is applied to a peroxidase containing solution and to a catalase containing solution.
- a suitable peroxidase is one producible by plants 20 (e.g. horseradish peroxidase) or microorganisms such as fungi or bacteria.
- the peroxidase is derived from Coprinus, e.g. C. cinereus or C. acrorhizus, or from Bacillus, e.g. B. pumilus, particularly a peroxidase according to International Patent Application WO 91/05858.
- the enzyme may furthermore be one which is produ ⁇ proficient by a method comprising cultivating a host cell transfor ⁇ med with a recombinant DNA vector which carries a DNA sequence encoding said enzyme as well as DNA sequences encoding func ⁇ tions permitting the expression of the DNA sequence encoding 0 the enzyme in a culture medium under conditions permitting the expression of the enzyme and recovering the enzyme from the culture.
- a recombinantly produced peroxidase is a peroxidase derived from a Coprinus sp., in particular ⁇ 5 macrorhizus or C. cinereus according to WO 92/16634.
- Catalases are known both from animal sources (e.g. cow liver) and from many different microorganisms.
- JP Patent Application 2-76579 discloses catalase from Aspercrillus ni ⁇ er strain NFAG-2.
- GB Patent No. 2,216,149 discloses catalase from Penicillium.
- the catalase is obtained from strains of Scvtalidium and Humicola as described in WO 92/17571.
- the enzyme is a protease.
- Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically or genetical- ly modified mutants are included. It may be a serine protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, e.g. subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279) .
- Examples of commer ⁇ cial Bacillus subtilisins are Alcalase ® , Savinase ® ' Esperase ® and Durazym" products of Novo Nordisk A/S.
- Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270.
- the enzyme is a lipase. Suitable lipases include those of bacterial and fungal origin. Chemical ⁇ ly or genetically modified mutants are included.
- lipase obtainable from Pseudomonas, Candida and Mucor, in particular Pseudomonas cepaciae, as described in EP 0 214 761, or lipase obtained by cloning the gene from Humicola lanuqinosa and expressing the gene in Asper ⁇ illus oryzae as described in EP 0 258 068, available under the trade mark Lipolase ® from Novo Nordisk A/S.
- the enzyme is an amylase. Suitable amylases include those of bacterial and fungal origin. Chemi ⁇ cally or genetically modified mutants are included.
- Amylases include, for example, ⁇ -amylases obtained from Bacillus, e.g. B. licheniformis, described in more detail in British Patent Specification No. 1,296,839.
- the enzyme is a cellulase.
- Suitable cellulases include those of bacterial and fungal origin, in particular cellulases from fungi are preferred. Chemically or genetically modified mutants are included. Examples of fungi which may be used for production of suitable cellulases are: Trichoderma, Phanerochaete, Humicola, Fusarium and Myceliop- thora.
- the method of invention further comprises addition of a water soluble polymer.
- Preferred water soluble polymers are glycols and amines, e.g. polyamines. More preferred are polyethylene glycol and polypropylene glycol . Most preferred are polyethylene glycols with a MW of from 200 to 10000. Water soluble polymers may be added in con ⁇ centrations of 1-50% w/w, preferably 2-40% w/w.
- the method may ad ⁇ ditionally comprise adding a salt in a concentration of up to 1.5 Molar, preferably in a concentration of up to 1.0 Molar.
- Preferred salts are salts of Magnesium, Calcium,
- Sodium, Potassium and Ammonium are preferred.
- the pH of the con- centrated aqueous solution, to which a water soluble polymer has been added may be adjusted to the optimum of the crystal ⁇ lization; in some cases optimum of the crystallization is at a level around pi of the enzyme.
- the acid may be inorganic or organic. Some examples are hydrochloric acid, sulfuric acid, nitrous acid, phosphoric acid, acetic acid, citric acid, and formic acid. Preferred acids are formic acid, citric acid, and acetic acid.
- a pre- ferred base is sodium hydroxide.
- salt is added to the aqueous mixture of proteins in a concentration of up to 1.5 Molar, preferably in a concentration of up to 1.0 Molar, and the pH of the solution is adjusted to the optimum of the crystallization.
- the method of invention causes the protein, in 5 particular the enzyme, to precipitate on crystalline form.
- Recovery of the crystalline protein may be accomplished by conventional methods, e.g. by filtration and subsequent drying, or by redissolution of the crystals for the manufacture of liquid protein products.
- Polyethylene glycol (PEG 4000) and CaCl 2 ;2H 2 0 were added in various amounts (cf. Fig. 1) , and pH was adjusted to pH 4.0 by addition of formic acid. The solution was stirred at 2528°C for 24 hours.
- the culture broth was subjected to 20 solid/liquid separation by centrifugation. Subsequently several ultrafiltrates containing various concentrations of peroxidase protein and other enzymes were obtained.
- the culture broth was subjected to solid/liquid separation by flocculation and filtration. Subsequently the filtrate was concentrated by evaporation and ultra-/diafiltration.
- the concentrate contained 1.2% w/w DS (dry substance) catalase protein and 5.5% w/w total dry substance.
- Polyethylene glycol (PEG 4000) and CaCl 2 ;2H 2 0 were added in various amounts (cf. Fig. 4) , and pH was adjusted to pH 6.8. The solution was stirred at 28°C for 48 hours.
- the crystallization yields at various CaCl 2 concentrations and at various PEG 4000 - concentrations are shown in the appended Fig. 4.
- the culture broth was subjected to solid/liquid separation by flocculation and filtration.
- the concentrate contained 0.28% w/w DS (dry substance) catalase protein and 9.2% w/w total dry substance.
- Polyethylene glycol (PEG 4000) and CaCl 2 ;2H 2 0 were added in various amounts (cf. Fig. 5), and pH was adjusted to pH 6.0 - 6.5. The solution was stirred at 28°C for 48 hours.
- the culture broth was subjected to solid/liquid separation by flocculation and filtration.
- the concentrate contained 0.28% w/w DS (dry substance) catalase protein and 9.2% w/w total dry substance.
- Polyethylene glycol PEG 1500 and CaCl 2 ;2H 2 0 were added in various amounts (cf. Fig. 6) , and pH was adjusted to pH 6.5 - 7.0. The solution was stirred at 28°C for 48 hours.
- the culture broth was subjected to solid/liquid separation by flocculation and centrifugation. Subsequently the supernatant was concentrated by ultrafiltra- tion. The concentrate contained 9.2% w/w DS (dry substance) protease protein and 19.6% w/w total dry substance. The conductivity was 1.55 mS/cm.
- Polyethylene glycol (PEG 4000) was added in various amounts (cf. Fig. 7) , and pH was adjusted to pH to 5.6. The solution was stirred at 28°C for 24 hours.
- the culture broth was subjected to solid/liquid separation by flocculation and centrifugation. Subsequently the supernatant was concentrated by ultrafiltra- tion. The concentrate contained 9.2% w/w DS (dry substance) protease protein and 19.6% w/w total dry substance. The conductivity was 1.55 mS/cm.
- Polyethylene glycol (PEG 4000) was added in various amounts together with 1.1% w/w CaCl 2 ;2H 2 0, and pH was adjusted to pH 5.6. The solution was stirred at 28°C for 24 hours. The crystallization yields at the various PEG concentrations are shown in the appended Fig. 8.
- Humicola insolens Cellulase obtained as described in WO 91/17244, was produced by fermentation and recovered by filtration, ultrafiltration and diafiltration with ionexchanged water.
- the concentrated cellulase solution had an enzyme concentration of 105 g/1, an enzyme purity of 58% of the dry matter content and a protein purity of 88%. pH was 7.0 and the conductivity was 607 ⁇ S .
- 1 peroxidase unit is the amount of enzyme that catalyzes the conversion of 1 ⁇ mole hydrogen peroxide per minute at the following analyti ⁇ cal conditions: 2.0 mM hydrogen peroxide, 0.46 mM 2,2'-azino- bis(3-ethylbenzothiazoline-6-sulfonate) , 0.086 M phosphate buffer, pH 7.0, incubated at 40°C, photometrically followed at 418 nm.
- 1 KPOXU 1000 POXU.
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- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Peptides Or Proteins (AREA)
- Separation Using Semi-Permeable Membranes (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Plant Substances (AREA)
- Electrical Discharge Machining, Electrochemical Machining, And Combined Machining (AREA)
Abstract
Description
Claims
Priority Applications (9)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE69429172T DE69429172T2 (en) | 1993-07-09 | 1994-06-23 | SEPARATION OF PROTEINS |
JP7503762A JPH08512294A (en) | 1993-07-09 | 1994-06-23 | Protein separation |
BR9407047A BR9407047A (en) | 1993-07-09 | 1994-06-23 | Process for separating a protein from an aqueous protein solution |
AT94920412T ATE209214T1 (en) | 1993-07-09 | 1994-06-23 | SEPARATION OF PROTEINS |
DK94920412T DK0707594T3 (en) | 1993-07-09 | 1994-06-23 | Separation of proteins |
US08/557,056 US5728559A (en) | 1993-07-09 | 1994-06-23 | Separation of proteins |
EP94920412A EP0707594B1 (en) | 1993-07-09 | 1994-06-23 | Separation of proteins |
AU71214/94A AU7121494A (en) | 1993-07-09 | 1994-06-23 | Separation of proteins |
FI960073A FI112949B (en) | 1993-07-09 | 1996-01-08 | Separation of proteins |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DK93830A DK83093D0 (en) | 1993-07-09 | 1993-07-09 | COURSE OF ACTION |
DK0830/93 | 1993-07-09 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1995001989A1 true WO1995001989A1 (en) | 1995-01-19 |
Family
ID=8098038
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/DK1994/000256 WO1995001989A1 (en) | 1993-07-09 | 1994-06-23 | Separation of proteins |
Country Status (13)
Country | Link |
---|---|
US (1) | US5728559A (en) |
EP (1) | EP0707594B1 (en) |
JP (1) | JPH08512294A (en) |
CN (1) | CN1054159C (en) |
AT (1) | ATE209214T1 (en) |
AU (1) | AU7121494A (en) |
BR (1) | BR9407047A (en) |
DE (1) | DE69429172T2 (en) |
DK (2) | DK83093D0 (en) |
ES (1) | ES2168300T3 (en) |
FI (1) | FI112949B (en) |
PT (1) | PT707594E (en) |
WO (1) | WO1995001989A1 (en) |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997032894A1 (en) * | 1996-03-08 | 1997-09-12 | Novo Nordisk A/S | Crystallization of a protein with a sulphur salt |
WO1997033905A1 (en) * | 1996-03-14 | 1997-09-18 | Novo Nordisk A/S | Increased yields of a crystallized protein by using a solid adsorption material |
WO1997033983A1 (en) * | 1996-03-11 | 1997-09-18 | Genencor International, Inc. | Crystalline protease and method for producing same |
WO1997034919A1 (en) * | 1996-03-15 | 1997-09-25 | Novo Nordisk A/S | Method for purification of a protein from a protein containing solution |
US6031082A (en) * | 1996-03-14 | 2000-02-29 | Novo Nordisk A/S | Increased yields of a crystallized protein by using a solid adsorption material |
WO2003050274A3 (en) * | 2001-12-11 | 2003-12-11 | Novozymes As | Process for harvesting crystalline particles from fermentation broth |
US7118891B2 (en) | 2001-12-11 | 2006-10-10 | Novozymes A/S | Crystal harvest from fermentation broth |
WO2009156073A1 (en) * | 2008-06-23 | 2009-12-30 | Bayer Technology Services Gmbh | Method for the selective separation of peptides and proteins by means of a crystallization process |
EP2125865B1 (en) | 2007-03-15 | 2014-07-30 | Novozymes A/S | Solubilization of protease crystals in fermentation broth |
EP2758514B1 (en) | 2011-09-22 | 2017-02-22 | Danisco US Inc. | Endogenous dnase activity to reduce dna content |
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US7250305B2 (en) * | 2001-07-30 | 2007-07-31 | Uab Research Foundation | Use of dye to distinguish salt and protein crystals under microcrystallization conditions |
US7247490B2 (en) * | 1999-04-06 | 2007-07-24 | Uab Research Foundation | Method for screening crystallization conditions in solution crystal growth |
US20030022383A1 (en) * | 1999-04-06 | 2003-01-30 | Uab Research Foundation | Method for screening crystallization conditions in solution crystal growth |
US7214540B2 (en) * | 1999-04-06 | 2007-05-08 | Uab Research Foundation | Method for screening crystallization conditions in solution crystal growth |
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ATE357656T1 (en) * | 1999-04-06 | 2007-04-15 | Univ Alabama Res Found | DEVICE FOR SCREENING CRYSTALIZATION CONDITIONS IN CRYSTAL GROWING SOLUTIONS |
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US7670429B2 (en) * | 2001-04-05 | 2010-03-02 | The California Institute Of Technology | High throughput screening of crystallization of materials |
US20070026528A1 (en) * | 2002-05-30 | 2007-02-01 | Delucas Lawrence J | Method for screening crystallization conditions in solution crystal growth |
AU2003256469A1 (en) * | 2002-07-10 | 2004-01-23 | Uab Research Foundation | Method for distinguishing between biomolecule and non-biomolecule crystals |
US20040185449A1 (en) * | 2003-03-20 | 2004-09-23 | Quinn John J. | Method for preparing assay samples |
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US7507552B1 (en) | 2004-04-16 | 2009-03-24 | Takeda San Diego, Inc. | Crystallization of histone deacetylase 2 |
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US20090043264A1 (en) * | 2005-04-24 | 2009-02-12 | Novo Nordisk A/S | Injection Device |
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JP5183499B2 (en) * | 2006-03-10 | 2013-04-17 | ノボ・ノルデイスク・エー/エス | Injection device |
EP2004671B1 (en) * | 2006-03-30 | 2015-12-09 | Novo Nordisk A/S | Two-phase precipitation of proteins |
DE602007004972D1 (en) | 2006-05-16 | 2010-04-08 | Novo Nordisk As | GEARING MECHANISM FOR AN INJECTION DEVICE |
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FR2902799B1 (en) | 2006-06-27 | 2012-10-26 | Millipore Corp | METHOD AND UNIT FOR PREPARING A SAMPLE FOR THE MICROBIOLOGICAL ANALYSIS OF A LIQUID |
US8163886B2 (en) | 2006-12-21 | 2012-04-24 | Emd Millipore Corporation | Purification of proteins |
US8569464B2 (en) | 2006-12-21 | 2013-10-29 | Emd Millipore Corporation | Purification of proteins |
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EP2125081B1 (en) * | 2007-03-23 | 2017-12-20 | Novo Nordisk A/S | An injection device comprising a locking nut |
EP2370561B1 (en) | 2008-12-16 | 2019-08-07 | EMD Millipore Corporation | Stirred tank reactor and method |
US8691918B2 (en) | 2010-05-17 | 2014-04-08 | Emd Millipore Corporation | Stimulus responsive polymers for the purification of biomolecules |
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US4400471A (en) * | 1980-11-03 | 1983-08-23 | Johal Sarjit S | Preparation and crystallization of Fraction I protein from plant sources |
US5244800A (en) * | 1990-04-27 | 1993-09-14 | The Uab Research Foundation | Crystals of human complement factor d that are triclinic |
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US4340676A (en) * | 1980-09-24 | 1982-07-20 | University Patents, Inc. | Method of crystallizing ribulose, 1,5-bisphosphate carboxylase/oxygenase from photosynthetic organisms, particularly plant leaves |
-
1993
- 1993-07-09 DK DK93830A patent/DK83093D0/en not_active Application Discontinuation
-
1994
- 1994-06-23 EP EP94920412A patent/EP0707594B1/en not_active Expired - Lifetime
- 1994-06-23 ES ES94920412T patent/ES2168300T3/en not_active Expired - Lifetime
- 1994-06-23 JP JP7503762A patent/JPH08512294A/en not_active Ceased
- 1994-06-23 CN CN94192657A patent/CN1054159C/en not_active Expired - Fee Related
- 1994-06-23 PT PT94920412T patent/PT707594E/en unknown
- 1994-06-23 AU AU71214/94A patent/AU7121494A/en not_active Abandoned
- 1994-06-23 DK DK94920412T patent/DK0707594T3/en active
- 1994-06-23 US US08/557,056 patent/US5728559A/en not_active Expired - Lifetime
- 1994-06-23 WO PCT/DK1994/000256 patent/WO1995001989A1/en active IP Right Grant
- 1994-06-23 AT AT94920412T patent/ATE209214T1/en not_active IP Right Cessation
- 1994-06-23 DE DE69429172T patent/DE69429172T2/en not_active Expired - Fee Related
- 1994-06-23 BR BR9407047A patent/BR9407047A/en not_active IP Right Cessation
-
1996
- 1996-01-08 FI FI960073A patent/FI112949B/en active
Patent Citations (2)
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US4400471A (en) * | 1980-11-03 | 1983-08-23 | Johal Sarjit S | Preparation and crystallization of Fraction I protein from plant sources |
US5244800A (en) * | 1990-04-27 | 1993-09-14 | The Uab Research Foundation | Crystals of human complement factor d that are triclinic |
Non-Patent Citations (1)
Title |
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EUR. J. BIOCHEM., Volume 189, 1990, ALEXANDER MCPHERSON: "Current approaches to macromolecular crystallization", page 1 - page 23, see pp. 12-13. * |
Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997032894A1 (en) * | 1996-03-08 | 1997-09-12 | Novo Nordisk A/S | Crystallization of a protein with a sulphur salt |
US6066481A (en) * | 1996-03-08 | 2000-05-23 | Novo Nordisk A/S | Crystallization of a protein with a sulphur salt |
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US6087148A (en) * | 1996-03-15 | 2000-07-11 | Novo Nordisk A/S | Method of purification of cellulose from a broth solution by crystallization |
WO1997034919A1 (en) * | 1996-03-15 | 1997-09-25 | Novo Nordisk A/S | Method for purification of a protein from a protein containing solution |
WO2003050274A3 (en) * | 2001-12-11 | 2003-12-11 | Novozymes As | Process for harvesting crystalline particles from fermentation broth |
US7118891B2 (en) | 2001-12-11 | 2006-10-10 | Novozymes A/S | Crystal harvest from fermentation broth |
EP2125865B1 (en) | 2007-03-15 | 2014-07-30 | Novozymes A/S | Solubilization of protease crystals in fermentation broth |
WO2009156073A1 (en) * | 2008-06-23 | 2009-12-30 | Bayer Technology Services Gmbh | Method for the selective separation of peptides and proteins by means of a crystallization process |
EP2758514B1 (en) | 2011-09-22 | 2017-02-22 | Danisco US Inc. | Endogenous dnase activity to reduce dna content |
Also Published As
Publication number | Publication date |
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FI960073A (en) | 1996-01-08 |
ATE209214T1 (en) | 2001-12-15 |
CN1126477A (en) | 1996-07-10 |
BR9407047A (en) | 1996-08-13 |
DK83093D0 (en) | 1993-07-09 |
EP0707594B1 (en) | 2001-11-21 |
US5728559A (en) | 1998-03-17 |
FI960073A0 (en) | 1996-01-08 |
AU7121494A (en) | 1995-02-06 |
ES2168300T3 (en) | 2002-06-16 |
DE69429172D1 (en) | 2002-01-03 |
CN1054159C (en) | 2000-07-05 |
FI112949B (en) | 2004-02-13 |
PT707594E (en) | 2002-05-31 |
JPH08512294A (en) | 1996-12-24 |
EP0707594A1 (en) | 1996-04-24 |
DE69429172T2 (en) | 2002-07-11 |
DK0707594T3 (en) | 2002-05-21 |
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