IE904254A1 - Process for the purification of lipocortins - Google Patents

Abstract

A process for obtaining lipocortins is described and entails a solution which contains lipocortins, especially PP4-X, PAP III, p68, anchorin II or lipocortin I or II, being contacted with a carrier-bound polysulphate of a saccharide or with a carrier-bound sulphated sugar, and the lipocortins being obtained.

Description

BEHRINGWERKE AKTIENGESELLSCHAFT HOE 89/B 043 - Ma 801 Dr.Ha/Sd Description Process for the purification of lipocortins The invention relates to a process for the purification of lipocortins, especially the proteins PP4-X, PAP III, p68, anchorin II and the 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 organisms, 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 neutralize these.

The previous processes for the rapid isolation of these proteins (Tait, J.F. et al. (1988) Biochemistry 27, 626820 6276) are not suitable for an industrial isolation of these 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 lipocortins<’may be carried out in a column or else in a batch here. - 2 The invention relates to a process for the isolation of lipocortins, which comprises bringing into contact in the presence of calcium ions a solution 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 supernatant 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/1 calcium ions, for example in the form of the chloride, acetate or lactate, 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 heparin, dextran sulfate, heparan sulfate, chondroitin sulfate, keratan sulfate or dermatan sulfate, the supernatant 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/1 calcium, and the lipocortin is eluted by means of a linear NaCl, LiCl or KC1 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 glycol, 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(sulfuric 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 functionalities have been introduced by means of carbodiimide.

In a preferred procedure, a solution containing a lipocortin, 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/1 calcium at a pH of 6-9, the mixture is incubated at 4*-30 °C for 3-300 min, the supernatant solution is separated off from the carrier resin, the adsorbent is washed, if appropriate, with a solution which has a pH of 6-9 and contains 0.00020.03 mol/1 calcium, and the proteins are eluted individually 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/1 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 poly(sulfuric 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 saccharide or by means of a sulfated sugar in the absence of calcium or in the presence of a chelateforming reagent such as EDTA, EGTA or a salt of citric acid or oxalic acid. 3. Ion exchange chromatography, for example on carboxymethyl-, DEAE-, QAE-RSepharose, RSephacel or cellulose (from Pharmacia, Sweden).

The process according to the invention is distinguished 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. - 4 For the large-scale isolation of these lipocortins, human placenta which is available in sufficient amounts and contains these proteins in high amounts is, above all, suitable as a source of raw material. For the purification 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,Ν,Ν',Ν'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 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 extracted with 50 1 of a solution of 0.02 mol/1 tris/HCl, pH 7.5, 0.15 mol/1 NaCl and 0.1 M tri-Na citrate, ammonium sulfate (33 % saturation) was added and the mixture was incubated with 4 1 of phenylsepharose in a batch process. After washing the resin, 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/1 tris/HCl, pH 8.0 (buffer A) and dialyzed against the same buffer. After the addition of CaCl2 up to a final concentration of 0.05 mol/1, 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 supernatant solution was separated off. The adsorbent was then - 5 washed with buffer A with the addition of 0.005 mol/1 CaCl2 and was pre-eluted with 0.06 mol/1 NaCl. The proteins PP4-X, PAP III, p68 and lipocortin I and II were eluted together all at once with 0.8 mol/1 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/1 and then bringing into contact the protein-containing solution with 200 ml of a heparin-RSepharose equilibrated in 0.02 mol/1 tris/10 HCl, pH 8.0, 0.001 mol/1 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 (lipocortin II, heavy chain) and 10 kD (light chain). The individual proteins were isolated by means of ion exchange 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 as described in Example 1 and the adsorbent was then washed. PP4-X (0.35 mol/1 NaCl) and PAP III (0.48 mol/1 NaCl) were eluted separately by stepwise elution. It was possible to elute lipocortin I, II and p68 together using 0.8 mol/1 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/1 NaCl were further eluted by ion exchange chromatography: The solution containing p68, lipocortin I and II was dialyzed against buffer A and pumped onto a DEAE35 RSepharose column equilibrated with buffer A. Adsorbed protein p68 was eluted by means of a NaCl gradient. The - 6 proteins lipocortin I and II which were not adsorbed in this chromatography were dialyzed against a buffer of 0.01 mol/1 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 applying a linear salt gradient of 0-0.7 mol/1 NaCl. The lipocortin proteins thus isolated had a purity of >95 % and, in SDS-PAGE, exhibited the molecular weights de10 scribed in Example 1 in each case.

Claims (12)

1. Patent. Claims

1. A process for the isolation of lipocortins comprising bringing into contact in the presence of calcium ions a solution which contains a lipocortin or several lipocortins with a carrier-bound poly(sulfuric acid ester) of a saccharide or with a carrierbound sulfated sugar, separating off the supernatant solution, washing the loaded carrier material, if appropriate, and eluting the lipocortins individually, 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 separation 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/1 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. A process as claimed in claim 1, substantially as hereinbefore described and exemplified.

12. Lipocortins whenever isolated by a process claimed in a preceding claim.