CA2030850A1 - Process for the purification of lipocortins - Google Patents
Process for the purification of lipocortinsInfo
- Publication number
- CA2030850A1 CA2030850A1 CA002030850A CA2030850A CA2030850A1 CA 2030850 A1 CA2030850 A1 CA 2030850A1 CA 002030850 A CA002030850 A CA 002030850A CA 2030850 A CA2030850 A CA 2030850A CA 2030850 A1 CA2030850 A1 CA 2030850A1
- Authority
- CA
- Canada
- Prior art keywords
- lipocortins
- carrier
- lipocortin
- sulfate
- bound
- 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.)
- Abandoned
Links
- 102000000412 Annexin Human genes 0.000 title claims abstract description 33
- 108050008874 Annexin Proteins 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 26
- 238000000746 purification Methods 0.000 title description 5
- -1 poly(sulfuric acid ester Chemical class 0.000 claims abstract description 15
- 108090000656 Annexin A6 Proteins 0.000 claims abstract description 12
- 150000001720 carbohydrates Chemical class 0.000 claims abstract description 12
- 102000004145 Annexin A1 Human genes 0.000 claims abstract description 11
- 108090000663 Annexin A1 Proteins 0.000 claims abstract description 11
- 102000004149 Annexin A2 Human genes 0.000 claims abstract description 11
- 108090000668 Annexin A2 Proteins 0.000 claims abstract description 11
- 108090000669 Annexin A4 Proteins 0.000 claims abstract description 11
- 102100034612 Annexin A4 Human genes 0.000 claims abstract description 11
- 235000000346 sugar Nutrition 0.000 claims abstract description 11
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims abstract description 11
- 238000002955 isolation Methods 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 13
- 229940032330 sulfuric acid Drugs 0.000 claims description 10
- 239000000872 buffer Substances 0.000 claims description 9
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 7
- 150000003839 salts Chemical class 0.000 claims description 7
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims description 6
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 claims description 6
- 229910001424 calcium ion Inorganic materials 0.000 claims description 6
- 239000012876 carrier material Substances 0.000 claims description 6
- 239000010414 supernatant solution Substances 0.000 claims description 6
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 5
- 229920000669 heparin Polymers 0.000 claims description 5
- 229960002897 heparin Drugs 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 229960000633 dextran sulfate Drugs 0.000 claims description 4
- 238000011109 contamination Methods 0.000 claims description 3
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 claims description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 claims description 2
- 229920000936 Agarose Polymers 0.000 claims description 2
- 229920001287 Chondroitin sulfate Polymers 0.000 claims description 2
- 229920000045 Dermatan sulfate Polymers 0.000 claims description 2
- 229920002307 Dextran Polymers 0.000 claims description 2
- 229920002971 Heparan sulfate Polymers 0.000 claims description 2
- 229920000288 Keratan sulfate Polymers 0.000 claims description 2
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 claims description 2
- 229940059329 chondroitin sulfate Drugs 0.000 claims description 2
- 229920001577 copolymer Polymers 0.000 claims description 2
- AVJBPWGFOQAPRH-FWMKGIEWSA-L dermatan sulfate Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@H](OS([O-])(=O)=O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](C([O-])=O)O1 AVJBPWGFOQAPRH-FWMKGIEWSA-L 0.000 claims description 2
- 229940051593 dermatan sulfate Drugs 0.000 claims description 2
- 229960002086 dextran Drugs 0.000 claims description 2
- KXCLCNHUUKTANI-RBIYJLQWSA-N keratan Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@H](COS(O)(=O)=O)O[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@H](O[C@@H](O[C@H]3[C@H]([C@@H](COS(O)(=O)=O)O[C@@H](O)[C@@H]3O)O)[C@H](NC(C)=O)[C@H]2O)COS(O)(=O)=O)O[C@H](COS(O)(=O)=O)[C@@H]1O KXCLCNHUUKTANI-RBIYJLQWSA-N 0.000 claims description 2
- 235000006408 oxalic acid Nutrition 0.000 claims description 2
- WXZMFSXDPGVJKK-UHFFFAOYSA-N pentaerythritol Chemical compound OCC(CO)(CO)CO WXZMFSXDPGVJKK-UHFFFAOYSA-N 0.000 claims description 2
- 229920000642 polymer Polymers 0.000 claims description 2
- 238000001179 sorption measurement Methods 0.000 claims description 2
- 239000002202 Polyethylene glycol Substances 0.000 claims 1
- 229920002401 polyacrylamide Polymers 0.000 claims 1
- 229920001223 polyethylene glycol Polymers 0.000 claims 1
- 238000000926 separation method Methods 0.000 claims 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 24
- 102000004169 proteins and genes Human genes 0.000 description 18
- 108090000623 proteins and genes Proteins 0.000 description 18
- 239000011780 sodium chloride Substances 0.000 description 12
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 5
- 102100023408 KH domain-containing, RNA-binding, signal transduction-associated protein 1 Human genes 0.000 description 5
- 229960005069 calcium Drugs 0.000 description 5
- 229910052791 calcium Inorganic materials 0.000 description 5
- 235000001465 calcium Nutrition 0.000 description 5
- 239000011575 calcium Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000004587 chromatography analysis Methods 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 3
- 239000003463 adsorbent Substances 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 206010053567 Coagulopathies Diseases 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229940044197 ammonium sulfate Drugs 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 230000002429 anti-coagulating effect Effects 0.000 description 2
- 230000035602 clotting Effects 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 210000002826 placenta Anatomy 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- RAXXELZNTBOGNW-UHFFFAOYSA-N 1H-imidazole Chemical compound C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 1
- GXVUZYLYWKWJIM-UHFFFAOYSA-N 2-(2-aminoethoxy)ethanamine Chemical compound NCCOCCN GXVUZYLYWKWJIM-UHFFFAOYSA-N 0.000 description 1
- 125000000022 2-aminoethyl group Chemical group [H]C([*])([H])C([H])([H])N([H])[H] 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- 101710158668 Placental protein Proteins 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 102000008217 Pregnancy Proteins Human genes 0.000 description 1
- MZVQCMJNVPIDEA-UHFFFAOYSA-N [CH2]CN(CC)CC Chemical group [CH2]CN(CC)CC MZVQCMJNVPIDEA-UHFFFAOYSA-N 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 229940106135 cellulose Drugs 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 150000002118 epoxides Chemical class 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000003169 placental effect Effects 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4721—Lipocortins
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Analytical Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
BEHRINGWERKE AKTIENGESELLSCHAFT HOE 89/B 043 - Ma 801 Dr.Ha/Sd Abstract Process for the purificatîon of lipocortins A process for the isolation of lipocortins is described, wherein a solution which contains lipocortins, especially PP4-X, PAP III, p68, anchorin II or lipocortin I or II is brought into contact with a carrier-bound poly(sulfuric acid ester) of a saccharide or with a carrier-bound sulfated sugar, and the lipocortins are isolated.
Description
J i~ ~ .. 3 ~
BE~RINGWERRE: ~TIENGESE:LLSCHAFT HOE 89/E~ 043 -- Ma 801 Dr . Ha/Sd Description Process for the purification of lipocortins The invention relates to a process for the puxification of lipocortins, especially the proteins PP4-X, PAP III, p68, anchorin II and ~he lipocortins I and II.
These proteins belong to a family of proteins which is referred to as lipocortins, calpactins or annexins. They have been detected in many organlsms, and also in man, in most organs and cell types. The most important functional properties of these proteins, which are known so far, are an anti-inflammatory and anticoagulatory action. This protein family can influence inflammatory disorders and disturbances of clotting which are often associated therewith such as disseminated intravasal clotting (DIC) and diminish or even neutrali~e these.
The previous processes for the rapid isolation of these proteins (Tait, J.F. et al. (1988) Biochemistry 27, 6268-6276) are not suitable for an industrial isolation ofthese proteins.
The object of the invention therefore was to develop a process for the purification of the proteins PP4-X, PAP
III, p68, anchorin II and lipocortins I and II.
Surprisingly, it has now been found that these proteins in the presence of calcium have affinity to poly(sulfuric acid esters) of saccharides or to sulfated sugars and that it is possible to bind them to carrier-bound poly-~sulfuric acid esters) of saccharides or to sulfated sugars during which procedure most of the contaminating proteins are not adsorbed. The adsorption of the lipo~
cortins may be carried out in a column or else in a batch here.
:
The invention relates to a process for the isolation of lipocortins, which comprises brinqing into contact in the presence of calcium ions a solu~ion which contains a lipocortin or several lipocortins with a carrier-bound poly(sulfuric acid ester) of a saccharide or with a carrier-bound sulfated sugar, separating off the super natant solution, washing the loaded carrier material, if appropriate, and eluting the lipocortins individually, in groups or totally by increasing the ionic strength.
In one procedure, a solution which contains 0.01-50 mg of a lipocortin/ml and 0~0001-0.1 mol/l calcium ions, for example in the foxm of the chloride, acetate or lactater and has a pH of 5.5-9.5 is brought into contact with a carrier-bound poly(sulfuric acid ester) of a saccharide or of a sulfated sugar (for example carrier-bound h~pa-rin, dextran sulfate~ heparan sulfate~ chondroitin sulfate, keratan sulfate or dermatan sulfate, the super-natant solution is separated off, the loaded carrier material is washed, if appropriate, with a buffer which has a pH of 5.5-9.5 and contains 0.0001-0.1 mol/l cal-cium, and the lipocortin is eluted by means of a linear NaCl, hiCl or KCl gradient.
If there is a mixture of lipocortins in solution, it is also possible to elute them in groups or together.
For example, insoluble dextran, agarose, acrylamide, a polymer into which amino functionalities have been introduced and/or copolymers of polyethylene ~lycol, pentaerythrite and methacrylate, or else a combination thereof can be used as a water-insoluble carrier matrix to which a poly(sulfuric acid ester) of a saccharide, or a sulfated sugar can be covalently bound. The poly(sul-furic acid ester) of saccharides or the sulfated sugar can be coupled by known methods, for example to a carrier material which has been preactivated by means of CNBr or epoxide or to a carrier material in which amino function-alities have been introduced by means of carbodiLmide.
` ' ' .', ' ~, ' ,:
.
.
.~.J S~
~ 3 --In a preferred procedure, a solution containing a lipo-cortin, or a solution which contains a combination of these proteins or all of these proteins is brought into contact with carrier-bound heparin or dextran sulfate, but preferably with a heparin affinity resin, in the presence of 0.0002-0.03 mol/l calcium at a pH of 6-9, the mixture is incubated at 4-30C for 3-300 min, the supernatant solution is separated off from ~he carrier resin, the adsorbent is washed, i appropriate, with a solution which has a pH of 6-9 and contains 0.0002-0.03 moltl calcium, and the proteins are eluted individu-ally by increasing the conductivity, preferably by means of a linear salt gradient, or in groups via stepwise elution or totally, preferably by increasing the salt concentration to 0.6-1.0 mol/l by means of NaCl, KCl, LiCl or a salt of citric acid.
If necessary, a second purification of individual or all lipocortins can follow. There are several possibilities for this:
1. Rechromatography on another carrier-bound Polyt 6ul-furic acid ester) of a saccharide or on another sulfated sugar.
BE~RINGWERRE: ~TIENGESE:LLSCHAFT HOE 89/E~ 043 -- Ma 801 Dr . Ha/Sd Description Process for the purification of lipocortins The invention relates to a process for the puxification of lipocortins, especially the proteins PP4-X, PAP III, p68, anchorin II and ~he lipocortins I and II.
These proteins belong to a family of proteins which is referred to as lipocortins, calpactins or annexins. They have been detected in many organlsms, and also in man, in most organs and cell types. The most important functional properties of these proteins, which are known so far, are an anti-inflammatory and anticoagulatory action. This protein family can influence inflammatory disorders and disturbances of clotting which are often associated therewith such as disseminated intravasal clotting (DIC) and diminish or even neutrali~e these.
The previous processes for the rapid isolation of these proteins (Tait, J.F. et al. (1988) Biochemistry 27, 6268-6276) are not suitable for an industrial isolation ofthese proteins.
The object of the invention therefore was to develop a process for the purification of the proteins PP4-X, PAP
III, p68, anchorin II and lipocortins I and II.
Surprisingly, it has now been found that these proteins in the presence of calcium have affinity to poly(sulfuric acid esters) of saccharides or to sulfated sugars and that it is possible to bind them to carrier-bound poly-~sulfuric acid esters) of saccharides or to sulfated sugars during which procedure most of the contaminating proteins are not adsorbed. The adsorption of the lipo~
cortins may be carried out in a column or else in a batch here.
:
The invention relates to a process for the isolation of lipocortins, which comprises brinqing into contact in the presence of calcium ions a solu~ion which contains a lipocortin or several lipocortins with a carrier-bound poly(sulfuric acid ester) of a saccharide or with a carrier-bound sulfated sugar, separating off the super natant solution, washing the loaded carrier material, if appropriate, and eluting the lipocortins individually, in groups or totally by increasing the ionic strength.
In one procedure, a solution which contains 0.01-50 mg of a lipocortin/ml and 0~0001-0.1 mol/l calcium ions, for example in the foxm of the chloride, acetate or lactater and has a pH of 5.5-9.5 is brought into contact with a carrier-bound poly(sulfuric acid ester) of a saccharide or of a sulfated sugar (for example carrier-bound h~pa-rin, dextran sulfate~ heparan sulfate~ chondroitin sulfate, keratan sulfate or dermatan sulfate, the super-natant solution is separated off, the loaded carrier material is washed, if appropriate, with a buffer which has a pH of 5.5-9.5 and contains 0.0001-0.1 mol/l cal-cium, and the lipocortin is eluted by means of a linear NaCl, hiCl or KCl gradient.
If there is a mixture of lipocortins in solution, it is also possible to elute them in groups or together.
For example, insoluble dextran, agarose, acrylamide, a polymer into which amino functionalities have been introduced and/or copolymers of polyethylene ~lycol, pentaerythrite and methacrylate, or else a combination thereof can be used as a water-insoluble carrier matrix to which a poly(sulfuric acid ester) of a saccharide, or a sulfated sugar can be covalently bound. The poly(sul-furic acid ester) of saccharides or the sulfated sugar can be coupled by known methods, for example to a carrier material which has been preactivated by means of CNBr or epoxide or to a carrier material in which amino function-alities have been introduced by means of carbodiLmide.
` ' ' .', ' ~, ' ,:
.
.
.~.J S~
~ 3 --In a preferred procedure, a solution containing a lipo-cortin, or a solution which contains a combination of these proteins or all of these proteins is brought into contact with carrier-bound heparin or dextran sulfate, but preferably with a heparin affinity resin, in the presence of 0.0002-0.03 mol/l calcium at a pH of 6-9, the mixture is incubated at 4-30C for 3-300 min, the supernatant solution is separated off from ~he carrier resin, the adsorbent is washed, i appropriate, with a solution which has a pH of 6-9 and contains 0.0002-0.03 moltl calcium, and the proteins are eluted individu-ally by increasing the conductivity, preferably by means of a linear salt gradient, or in groups via stepwise elution or totally, preferably by increasing the salt concentration to 0.6-1.0 mol/l by means of NaCl, KCl, LiCl or a salt of citric acid.
If necessary, a second purification of individual or all lipocortins can follow. There are several possibilities for this:
1. Rechromatography on another carrier-bound Polyt 6ul-furic acid ester) of a saccharide or on another sulfated sugar.
2. Removal of the salt or the calcium ions, for example by dialysis, and chromatography by means of a carrier-bound poly(sulfuric acid ester) of a sac-charide or by means of a sulfated sugar in the absence of calcium or in the presence of a chelate-forming reagent such as EDTA, EG~A or a salt of citric acid or oxalic acid.
3. Ion exchanye chromatography, for example on c~rboxy-methyl-, DEAE-, QAE-RSepharose, RSephacel or cellu-lose ~from Pharmacia, Sweden).
The process according to the invention is di~inguished by the fact that it is possible, also on a large scale, to isolate the proteins PP4-X, PAP III, p68, anchorin II, and lipocortin I and II in a few steps at a very high purity and in a very high yield.
.
~ r~ ~ r~
The process according to the invention is di~inguished by the fact that it is possible, also on a large scale, to isolate the proteins PP4-X, PAP III, p68, anchorin II, and lipocortin I and II in a few steps at a very high purity and in a very high yield.
.
~ r~ ~ r~
- For the large-scale isolation of these lipocortins, human placenta which is a~ailable in sufficient ~nounts and contains these proteins in high amounts is, above alll suitable as a source of raw material. For th~ purifica-tion of anchorin II, above all the fibroblasts containing this protein are especially suitable.
Explanation of the abbreviations:
EDTA: ethylenediaminetetraacetic acid EGTA: ethylene glycol-bis(aminoethyl ether)-N,N,N',N'-tetraacetic acid DEAE: diethylaminoethyl QAE: quaternary aminoethyl PP4-X: placental protein PAP: placental anticoagulatory protein The following examples are intended to illustrate the invention:
Example 1 36 kg of ripe human placenta was comminuted, washed several times with 0.9 g/100 ml NaCl solution and then freeze-dried. The lyophilized product (3 kg) was ex-tracted with 50 1 of a solution of 0.02 mol/l tris~HC1, pH 7.5, 0.15 mol/l NaCl and 0.1 M tri-Na citrate, ammon-ium sulfate (33 % saturation) was added and the mixture was incubated with 4 1 of phenylsepharose in a batch ~5 process. After washing the re6in, the lipocortins were eluted all at once with water, precipitated by the addition of ammonium sulfate to 80 ~ saturation~ pelleted by centrifugation, taken up in a solution of 0.02 mol/l tris/HCl, pH 8.0 (buffer A) and dialyzed against the same buffer. After the addition o~ CaCl2 up to a final concent-ration of 0.05 mol/l, 1 1 of a heparin-RSepharose was added to the dialyzate in a batch process, the mixture was stirred at room temperature for 60 min and the super-natant solution was separated off. The adsorbent was then .-. .
.
;. , :
washed with buffer A with ~he addition of 0.005 mol/lCaCl2 and was pre-eluted with 0.06 mol/l NaCl. The proteins PP4-X, PAP III, p68 and lipocortin I and II were eluted together all at once with 0.8 mol/l NaCl.
After the dialysis of the eluate against buffer A, the remaining contaminations were removed by adding EDTA to a final concentration of 0.001 mol/l and then bringing into contact the protein-containing solution with 200 ml of a heparin-RSepharose equilibra~ed in 0.02 mol/l tris/-HCl, pH 8.0, 0.001 mol/l EDTA, stirring the mixture at room temperature for 60 min and separating off the supernatant solution containing the lipocortin proteins.
In SDS-polyacrylamide gel electrophoresis (PAGE), the lipocortin proteins purified in this manner exhibited bands with molecular weights of 33 kD (PP4-X), 34 kD (PAP
III), 68 kD (p68), 36 kD (lipocortin I) and 37 kD (lipo-cortin II, heavy chain) and 10 kD ~light chain). The individual proteins were isolated by means of ion ex-change chromatography as is described in Example 2.
The yields for the individual proteins were between 70 and 90 % in relation to the phenyl-RSepharose eluate.
Example 2 A solution containing PP4-X, PAP III, p68 and lipocortin I and II was brought into contact with heparin-RSepharose ~5 as described in ~xample 1 and the adsorbent was then washed. PP4-X (O.35 mol/l NaCl) and PAP III (O.48 mol/l NaCl) were eluted separately by stepwise elution. It was possible to elute lipocortin I, II and p68 together using O.8 mol/l NaCl. PP4-X, PAP III, or p68, lipocortin I and II were rendered free of remaining contaminations as described in Example 1. The proteins eluted by 0.8 mol/l NaCl were further eluted by ion exchange chromatography:
The solution containing p68, lipocortin I and II was dialyzed against buf fer A and pumped onto a DEAE-RSepharose column equilibrated with buffer A. Adsorbedprotein p68 was eluted by means of a NaCl gradient. The ' ) gj~ t~
proteins lipocortin I and II which were not adsorbed in this chromatography were dialyzed against a buffer of 0.01 mol/l imidazol/HCl, pH 6.0, brought into contact with carboxymethyl cellulose while stirring, and the adsorbance was then washed out and columns packed with it. Lipocortin I and II were eluted separately by apply-ing a linear salt gradient o~ 0-0.7 mol/l NaCl. The lipocortin proteins thus isola~ed had a purity of >95 ~
and, in SDS-PAGE, exhibited the molecular weights de-scribed in Example 1 in each case.
': ., :
, -. ~ , , - .
,
Explanation of the abbreviations:
EDTA: ethylenediaminetetraacetic acid EGTA: ethylene glycol-bis(aminoethyl ether)-N,N,N',N'-tetraacetic acid DEAE: diethylaminoethyl QAE: quaternary aminoethyl PP4-X: placental protein PAP: placental anticoagulatory protein The following examples are intended to illustrate the invention:
Example 1 36 kg of ripe human placenta was comminuted, washed several times with 0.9 g/100 ml NaCl solution and then freeze-dried. The lyophilized product (3 kg) was ex-tracted with 50 1 of a solution of 0.02 mol/l tris~HC1, pH 7.5, 0.15 mol/l NaCl and 0.1 M tri-Na citrate, ammon-ium sulfate (33 % saturation) was added and the mixture was incubated with 4 1 of phenylsepharose in a batch ~5 process. After washing the re6in, the lipocortins were eluted all at once with water, precipitated by the addition of ammonium sulfate to 80 ~ saturation~ pelleted by centrifugation, taken up in a solution of 0.02 mol/l tris/HCl, pH 8.0 (buffer A) and dialyzed against the same buffer. After the addition o~ CaCl2 up to a final concent-ration of 0.05 mol/l, 1 1 of a heparin-RSepharose was added to the dialyzate in a batch process, the mixture was stirred at room temperature for 60 min and the super-natant solution was separated off. The adsorbent was then .-. .
.
;. , :
washed with buffer A with ~he addition of 0.005 mol/lCaCl2 and was pre-eluted with 0.06 mol/l NaCl. The proteins PP4-X, PAP III, p68 and lipocortin I and II were eluted together all at once with 0.8 mol/l NaCl.
After the dialysis of the eluate against buffer A, the remaining contaminations were removed by adding EDTA to a final concentration of 0.001 mol/l and then bringing into contact the protein-containing solution with 200 ml of a heparin-RSepharose equilibra~ed in 0.02 mol/l tris/-HCl, pH 8.0, 0.001 mol/l EDTA, stirring the mixture at room temperature for 60 min and separating off the supernatant solution containing the lipocortin proteins.
In SDS-polyacrylamide gel electrophoresis (PAGE), the lipocortin proteins purified in this manner exhibited bands with molecular weights of 33 kD (PP4-X), 34 kD (PAP
III), 68 kD (p68), 36 kD (lipocortin I) and 37 kD (lipo-cortin II, heavy chain) and 10 kD ~light chain). The individual proteins were isolated by means of ion ex-change chromatography as is described in Example 2.
The yields for the individual proteins were between 70 and 90 % in relation to the phenyl-RSepharose eluate.
Example 2 A solution containing PP4-X, PAP III, p68 and lipocortin I and II was brought into contact with heparin-RSepharose ~5 as described in ~xample 1 and the adsorbent was then washed. PP4-X (O.35 mol/l NaCl) and PAP III (O.48 mol/l NaCl) were eluted separately by stepwise elution. It was possible to elute lipocortin I, II and p68 together using O.8 mol/l NaCl. PP4-X, PAP III, or p68, lipocortin I and II were rendered free of remaining contaminations as described in Example 1. The proteins eluted by 0.8 mol/l NaCl were further eluted by ion exchange chromatography:
The solution containing p68, lipocortin I and II was dialyzed against buf fer A and pumped onto a DEAE-RSepharose column equilibrated with buffer A. Adsorbedprotein p68 was eluted by means of a NaCl gradient. The ' ) gj~ t~
proteins lipocortin I and II which were not adsorbed in this chromatography were dialyzed against a buffer of 0.01 mol/l imidazol/HCl, pH 6.0, brought into contact with carboxymethyl cellulose while stirring, and the adsorbance was then washed out and columns packed with it. Lipocortin I and II were eluted separately by apply-ing a linear salt gradient o~ 0-0.7 mol/l NaCl. The lipocortin proteins thus isola~ed had a purity of >95 ~
and, in SDS-PAGE, exhibited the molecular weights de-scribed in Example 1 in each case.
': ., :
, -. ~ , , - .
,
Claims (11)
1. A process for the isolation of lipocortins compris-ing bringing into contact in the presence of calcium ions a solution which contains a lipocortin or several lipocortins with a carrier-bound poly(sul-furic acid ester) of a saccharide or with a carrier-bound sulfated sugar, separating off the supernatant solution, washing the loaded carrier material, if appropriate, and eluting the lipocortins individu-ally, in groups or totally by increasing the ionic strength.
2. The process as claimed in claim 1, wherein the lipocortins are PP4-X, PAP III, p68, anchorin II or lipocortin I or II.
3. The process as claimed in claim 1, wherein the lipocortins are subsequently purified by adsorption of remaining contaminations on a poly(sulfuric acid ester) of a saccharide or on a sulfated sugar in the absence of calcium ions or in the presence of an excess of a chelate-forming reagent, and by separa-tion of the lipocortin-containing solution.
4. The process as claimed in at least one of claims 1 and 3, wherein the carrier material is insoluble dextran, agarose, polyacrylamide, an insoluble polymer into which amino functionalities have been introduced or a copolymer of polyethylene glycol, pentaerythrite and methacrylate, or a combination thereof.
5. The process as claimed in at least one of claims 1 and 3, wherein the poly(sulfuric acid ester) of a saccharide is heparin, dextran sulfate, heparan sulfate, chondroitin sulfate, keratan sulfate or dermatan sulfate.
6. The process as claimed in at least one of claims 1 and 3, wherein carrier-bound heparin or dextran sulfate is used.
7. The process as claimed in at least one of claims 1 and 3, wherein carrier-bound heparin is used.
8. The process as claimed in claim 1, wherein the process is carried out in a buffer which has a pH of 5.5-9.5 and contains 0.001-0.1 mol/l calcium ions.
9. The process as claimed in claim 2, wherein EDTA, EGTA, a salt of citric acid or oxalic acid, or a combination thereof is used as chelate-forming reagent.
10. The process as claimed in claim 2, wherein EDTA or EGTA is used as chelate-forming reagent.
11. The process as claimed in claim 1 and substantially as described herein.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3939169A DE3939169A1 (en) | 1989-11-27 | 1989-11-27 | METHOD FOR PURIFYING LIPOCORTINES |
| DEP3939169.8 | 1989-11-27 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2030850A1 true CA2030850A1 (en) | 1991-05-28 |
Family
ID=6394271
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002030850A Abandoned CA2030850A1 (en) | 1989-11-27 | 1990-11-26 | Process for the purification of lipocortins |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP0430121A1 (en) |
| JP (1) | JPH03176500A (en) |
| KR (1) | KR910009727A (en) |
| AU (1) | AU6693590A (en) |
| CA (1) | CA2030850A1 (en) |
| DE (1) | DE3939169A1 (en) |
| IE (1) | IE904254A1 (en) |
| PT (1) | PT95992A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2542391A (en) * | 2015-09-17 | 2017-03-22 | Annexin Pharmaceuticals Ab | Process of manufacture |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3724726A1 (en) * | 1987-07-25 | 1989-02-02 | Behringwerke Ag | METHOD FOR PURIFYING THE PLACENTARY TISSUE PROTEIN PP4 |
| DE3737237A1 (en) * | 1987-11-03 | 1989-05-18 | Behringwerke Ag | ANTICOAGULATORY PROTEIN PP4-X, ITS PRODUCTION AND USE |
-
1989
- 1989-11-27 DE DE3939169A patent/DE3939169A1/en not_active Withdrawn
-
1990
- 1990-11-26 IE IE425490A patent/IE904254A1/en unknown
- 1990-11-26 KR KR1019900019187A patent/KR910009727A/en not_active Withdrawn
- 1990-11-26 PT PT95992A patent/PT95992A/en not_active Application Discontinuation
- 1990-11-26 CA CA002030850A patent/CA2030850A1/en not_active Abandoned
- 1990-11-26 AU AU66935/90A patent/AU6693590A/en not_active Abandoned
- 1990-11-26 JP JP2318172A patent/JPH03176500A/en active Pending
- 1990-11-26 EP EP90122493A patent/EP0430121A1/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| JPH03176500A (en) | 1991-07-31 |
| PT95992A (en) | 1991-09-13 |
| DE3939169A1 (en) | 1991-05-29 |
| IE904254A1 (en) | 1991-06-05 |
| KR910009727A (en) | 1991-06-28 |
| AU6693590A (en) | 1991-05-30 |
| EP0430121A1 (en) | 1991-06-05 |
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