IE912093A1 - Process for the separation of proteins or polypeptides - Google Patents

Abstract

Phosvitin or a modified phosvitin (for example by trypsin digestion) immobilised and coupled to a suitable matrix may be used for the separation and purification of proteins or polypeptides and in the removal of metal ions from biological material. If desired the phosvitin or modified phosvitin may be in the form of a metal chelate complex. This chelate may be used to remove peroxide from solvents.

Description

PROCESS FOR THE SEPARATION Of PROTBIHS. POLYPEPTIDES OR METALS The present invention relate· to separation of polypeptides or proteins or removal of metal ions, and to chromatographic agents suitable therefor.

Chromatographic agents for use in various separation processes are veil known in the art. However, there has been a tremendous need for chromatographic agents with a specific affinity for proteins. X few proteins such as lectins and protein A are used as ligands for affinity separation. However, their use is limited: lectins can be used only for the isolation and purification of glycoproteins, and protein A only for the isolation and purification of immunoglobulins.

The applicants have discovered that immobilised phosvitin and modified phosvitin act as excellent chromatographic agents for the separation of polypeptides and proteins, especially those that have a high affinity for phospho-serine clusters.

It is known that phosvitin has a high affinity for metal ions. The applicants have found also that this property is also exhibited by immobilised phosvitin, which can be utilised in e system working on the principles of metal-chelate chromatography. Metal ions such, for example, as Fe3+, Fe2*, Ca2+, Hg2+, Mn2+, Cu2+, :| :i I', co2+ and Zn2* are all biologically very important because of their involvement in a variety of catalytic processes Accordingly, the present invention provides the use of phosvitin or a modified phosvitin for the preparation of a chromatographic agent for the separation and/or 5 purification of polypeptides and proteins or for the removal of metal ions from biological material.

The present invention also provides phosvitin or a modified phosvitin immobilised and coupled to a suitable matrix, for use in the separation and/or purification of polypeptides and proteins.

The phosvitin or modified phosvitin aay have been produced by recombinant DNA technology, and the term phosvitin as used herein includes both molecules of natural origin and the corresponding recombinant molecules. In the case of a modified phosvitin, the modified molecule Itself may be produced by recombinant DNA technology, or recombinant phosvitin may ba produced and then modified. λ modified phosvitin may have, for example, one or more of the following modifications while still preserving binding ability: removal of some or all of the carbohydrate; removal of one or more amino acids; addition of one or more amino acids; modification at one or more individual amino acid residues; replacement of one or more individual amino acid residues, for example of aspartic acid by glutamic acid or lysine by arginine; appropriate physical change to the molecule.

If desired, the phosvitin or Modified phosvitin nay be in the font of a metal chelate complex.

The phosvitin or modified phosvitin may be used in the isolation of polypeptides and proteins, for the separation of various individual polypeptides and proteins from their impurities, and for the purification of polypeptides and proteins. Thue, for example, the chromatographic agent nay be used for the resolution of a mixture of proteins or polypeptides fron a broth or an extract, and/or it nay be used to obtain a protein or polypeptide in a substantially pure (homogeneous) form. λ non-chelated phosvitin or modified phosvitin nay also be used for the renoval of netal ions from biological material, especially fron physiological fluids, for example fron blood, serum or plasma.

The present invention further provides a process for the separation and/or purification of a polypeptide or a protein, or for the removal of netal ions from biological material, vhereln there is used ee chromatographic agent phosvitin or a modified phosvitin, immobilised and coupled to a suitable matrix.

Phosvitin is rich in phoephorylated serine residues and these normally occur in clusters in that protein.

The applicants have found hy examination of X-ray crystallography data, in relation to proteins having serine clusters, that the configuration of those clusters varies greatly from protein to protein, implying, they - 4 believe, that the amino acid· in proximity to the flusters dictate the configuration thoaa aerina clusters can assume.

The applicants have also discovered that proteins 5 such as, for example, cytochrome-C, lysozyme, EcoR^ and human Follicle Stimulating hormone (FSH) have an extremely high affinity for the phospho-serine residues of phosvitin in its immobilised form. These proteins such as cytoohrome-C and lysosyme carry charge clusters near their c-terminal regions, and the applicants believe that charge cluster regions which complement certain phospho-serine cluster domains on phosVitin are involved in the observed affinity phenomenon for these proteins.

Xt is considered that tha binding effects observed are not merely th· result of a generalised electrostatic interaction between the polyanionic phosvitin molecule end a polycationic protein or polypeptide. It ie believed that it ie the particular structure and configuration of the phosvitin molecule at and in the region of the phosphoserine clusters that leads to a specific interaction vith certain particular proteins and polypeptides that have a complementary structure and configuration at and in the region of charge clusters.

Accordingly, also, ve believe, suitable modified phosvitinm are especially those retaining the important phosphorylated serine residues, especially those in which some or all, preferably the majority, of the clusters of phoephorylated earIne residues are retained. Advantageously, substantially all such clusters are retained.

Further evidence for the postulated involvement of 5 domains in the interaction is given by experiments in which modifications of arginine residues in lysozyme with diacetyl and of lysine residues in cytochroaa-C with acetic anhydride were carried out. The applicants have found that these modifications lead to complete loss of the binding property of these proteins to a phosvitinSepharose matrix.

Accordingly, polypeptides end proteins that may be separated and/or purified by the non-chelated chromatographic agents according to the invention ere especially those having charge clusters, including, for example, various growth factors, DNA binding proteins (those involved in early gene replication and transcription processes) and DNA- and RNA-modifying enzymes. Examples include those given in the following Table 1: Table 1 A. Growth Hormones/Faotorai a) Adrenocorticotropic hormone b) Parathyroid hormone c) Fibroblast growth factors (both acidic a basic) d) Astroglial growth factors l a 2 e) Retina-derived growth factor t) Eye-derived growth factor g) Cartilage-derived growth factor h) Endothelial cell growth factor B. DNA binding protein·: a) Protein· having POO domains b) Proteins having Homeo domains c) zinc-finger proteins d) Leucine Zipper proteins e) Amphipathic helix-loop-helix motif-containing proteins C. DNA-modifying enzymes D. RNA-modify ing enzymes B. DNA-recombinant fusion protein products: Since certain domains in proteins such as cytochrome-c end lysozyme have strong affinity for phoevitin, it im considered that the engineering of the genes corresponding to those domains along with the gene coding for a protein of interest into an organism to produce a fusion protein containing those domains will facilitate their rapid and specific purification using the phosvitln/modifled phosvitin chromatographic agent according to the invention.

It has been proposed to use immobilised phoevitin to purify protein kinases, for which enzymes phosvitin can act as a substrate. Such use is, however, limited to that particular class of enzymes, and is not included in the present invention. ie 912093 - 7 The chemical and structural features of phosvitin in unmodified or modified form that make it eminently suitable for use as chromatographic ligand based, it is believed, on the principle Of charge cluster interactions also lend themselves to the generation of metal-chelates.

This ability to form metal chelates is of use, not only for removal of metals from biological and nonbiologioal materials, but also for generation of a metalohelate ahrematographie medium, and this may used in the separation and/or purification of both biological and non-biological material. Thus, ve believe that metalcontaining proteins and those having high affinity for metal ions may be purified by metal chelate chromatography.

Thus, for example, the applicants have found that a phoevitin-Sepharose matrix with appropriate chelation vith zinc results in a complex which exhibits affinity to proteins: for example it exhibited strong binding of at least two proteins from egg white (lio Kd and 120 Kd). similarly, chelate complexes with Pe3+, Pe2+, Cu2* and Ca2+ bound to phosvitin may be prepared and used for the purification of metal-dependant enzymes and other proteins, and an iron chelate complex of phosvitin affinity material may be used to remove peroxides from solvents.

The high affinity exhibited by phosvitin in unmodified or modified form towards metal ions indicates that the phosvitin matrix may also be used for scavenging excess metal ions froa physiological fluids, for example by haemodialysis. For example, in iron-overload states, the phosvitin matrix may be used, for example for iron scavenging from eerun in Cooley's anaemia.

Phosvitin is a naturelly-ooourring protein, found in avian and fish eggs. Phoavitin can be obtained in purified fora (electrophoretically homogeneous) by a number of techniques known per se. for example aa io described in J. Am. Chem. Soc. [1949] 71. 3670. Thue a chromatographic agent used in the present invention has the advantage that it utilises a protein which is naturally abundant and which can be purified vith relative ease.

Advantageously, the 8-form of phosvitin nay be used; this has a higher phosphate content than the a-fora.

The invention includes also the use of modified forms of phosvitin, whether or not prepared from phosvitin itself, and as well as the possibility of using a modified phosvitin Ah initio, the possibility of carrying out one or more modifications at any suitable stage in the preparation of the chromatographic agent may be mentioned.

Modified phosvitlns having affinity for metal ions, for example substantially the same affinity as has phosvitin, should especially be mentioned.

Modification to chain length may be carried out, for IE 912095 example, by chemical and/or enzymatic means, for example proteolysis with the protease trypsin. It is surprising that this proteolysis has proved possible, because it is generally considered that phosvitin ie resistant to proteolysis. Phosvitin modified in this way and iasobliisea on a suitable matrix can have an especially high binding capacity.

Other chemioal and/or enzymatic modifications directed at specific amino acid residues are also possible. Furthermore, if the gene for phosvitin is cloned into another organism to produce a recombinant phosvitin, site-directed-mutagenesis may be used to change a particular amino acid, tar example aspartic acid to glutamic acid or lysine to arginine. Such modifica15 tions are well-known in molecular biology. If a resulting recombinant phosvitin or modified phosvitin molecule is not already phoephorylated, a phosphorylation reaction should generally be carried out.

The phosvitin or phosvitin modified for example as above may be in the native glycosylated form, or it may be partly or fully deglycosylated. Daglycosylatlon methods are described in the literature. For removal of asparagine-linked (N-linked) glycosylated moieties see Tarentino A.L., Gomez G.M. and Plummer T.H., (1985), Biochemistry, 21, 4665-4671; for removal of serine-and threonine-linked (O-linked) glycosylated moieties see A.S.B. Edge, i£ fil, (1981), Annal Biochem, lie, 131-137.

Appropriate physical modification of the phosvitin should also be mentioned.

Two or more modifications may be carried out In any suitable order; for example, change of amino acid sequence and/or of chain length may he carried out before or after deglycosylation. Unless the context indicates otherwise, references herein to phosvitin* are used to include modified phosvitin.

The chromatographic agent used according to the 10 Invention may be prepared by methods known per mm: see, for example X new method for the analysis of blood serum glycoproteins using Sepharose coupled Lectins, S. Thompson and G.A. Turner, in Lectin·, edited by T.C. B0g-Hansen and D.L.J. Freed, pp 453-458, published by Sigma Chemical Company, 1988. The phosvitin may be attached directly to the matrix or Indirectly, by use of spacer arms.

Thus, a chromatographic agent comprising phosvitin or a modified phosvitin immobilized and coupled to a suitable matrix may be prepared by a process comprising mixing the phosvitin with the matrix in the presence of a buffer so that the pH of the mixture is in the range preferably of froa 8.0 to 8.3, washing away the excess ligand of the phosvitin and then blocking the remaining active groups of the matrix hy treating the mixture with an amine to produce ooupled phosvitin-matrix, washing the resulting product and recovering the coupled phosvitin11 matrix.

Th· matrix may be, for example, a Sepharose gel. Ne have used CXBr-activated sepharose and found that it efficiently couples and immobilises phosvitin and modified phosvitin. Activation of Sepharose with cyanogen bromide and coupling of proteins such as lectins to such activated matrix is veil known in the art. In addition to cyanogen bromide-activated-Sspharose, other support media/matrices, such, for example, ae agarose, acrylamide, silica and suitable fibres (both synthetic and natural) which are appropriately modified to enable coupling of proteins, may be used. The use of spacer arms usually employed for coupling containing β to 12 carbon atoms, for example 6-amlnohexanoic acid or 1,4bis(2,3-epoxypropxy)butane, to increase the binding capacities of the affinity material may provide an additional advantage.

The weight ratio of phosvitin to matrix used aay be, for example, substantially 0.005:1. The use of 6 to 10 mg of phosvitin per ml of swollen activated Sepharose or other matrix is recommended. The maximum amount of coupling we have observed is about 6ng/ml; this range of protein gives en affinity product of sufficient capacity for example for purification of proteins.

Mixing of tbe phosvitin and matrix may be carried out, for example, at a temperature in the range of froa to 25*C, more especially at substantially 4°C. - 12 The coupling of th* phoevitin to the matrix ie carried out in the presence of e suitable buffer. For activated matrices reactive to amine functions, this buffer should preferably be free of primary amines, since S if the coupling buffer contains reactive amino groups along with the protein ligand to be coupled, then the extent of protein ligand attachment to the matrix will be reduced, resulting in lower affinity capacity. Any buffer lacking an amino group may be employed, although most preferred are sodium bicarbonate and borate buffers. In general the buffer should provide a pH in the range of from 8.0 to 8.3. Excellent results may be obtained employing sodium bicarbonate buffer containing about 0.5 M NaCl. The use of a o.l M NbHC03 buffer having a pH of substantially 8.3 and containing 0.5 M NaCl should especially be mentioned.

After coupling with the matrix, excess ligands are washed away, for example with the buffer used for the coupling, and as soon as practicable thereafter the remaining active groups are blocked, generally with an amine or an amino acid. Primary amines are preferred. Bepecially good results have been obtained using ethanolamine. Amino acids may also be used to block excess reactive sites but are leee preferred ae these would introduce unwanted charges. The amine may be used, for example, in a concentration of from 0.1 to 1.0 K; l M ethanolamine is especially suitable.

The blocking reaction nay be carried out, for example, et a pH of from 7.5 to 9.5, more especially at a pH of substantially 9. It may be carried out, for example, for a period of from 2 to 18 hours, for example for substantially 16 hours. Suitably a temperature in the range of from 4 to 25*c, for example substantially 4’C, is used. Blocking vith 1 M ethanolamine at a pH of 9 for 16 hours at a temperature of 4‘C should especially be mentioned.

The resulting coupled phosvitin-matrix is then generally washed, to remove non-specifically bound proteins, if any, from the matrix. Usually, two different pHs are used, more especially two or more cycles of alternating pH. The number of cycles is generally three, although the number of such cycles is not critical.

Tbree washing cycles, for example, may be carried out, each cycle consisting, for example, of ο,ΐ M acetate buffer pH 4.0 containing 0.5 M NaCl, followed by a wash with 0.1 M Tris-HCl pH 8.0 containing 0.5 H NaCl. The applicants have found that three such washes generally remove non-specifically bound proteins (if any), from the matrix, as determined by absorbance at 280nm. Tbe chromatographic agent may then be recovered by filtration. Washing and recovery procedures used in coupling proteins to activated matrices are well known.

Preparation of a chromatography column may than be carried out by known methods. For example, after packing a suitable column vith the phosvitin matrix, equilibration may bo carried out for example with Tris HCl buffer, suitably vith 10 to 50 ott Tris HCl, pH 7.5 to 8.5; a suitable flow rate is 0.25 ml/min.

To prepare a metal chelate, the column after equilibration may be treated with a suitable buffer containing metal ions; usually the same buffer used for equilibration is used in thie step, for example lOmH Trim buffer, pH 7.5, containing, for example, 0.1 M zinc acetate, 0.1 M ferric chloride or 0.1 H calcium chloride. Two to three column volumes of such metal salt-containing buffer is suitably passed through the column, and the column then equilibrated with buffer alone.

Preparation of beads carrying a metal chelate, for example an iron chelate, for example for peroxide removal from solventa, should also be mentioned. Methods for the production of such beads are described in the literature.

The present invention also provides a chromato20 graphic agent vhich comprises phosvitin in the form of a metal chelate complex, immobilised and coupled to a suitable matrix, and a chromatographic agent which comprises a modified phosvitin, if desired in the form of a metal chelate complex, immobilised and coupled to a suitable matrix, and a process for their preparation ae described above.

The actual process for the separation or - 15 purification of proteina/polypeptides or for the removal of metal ions nay be carried out according to methods known per sef using set protocols, for example as follows. λ column ie prepared and equilibrated as described above. The column is then loaded with the crude material (containing for example 1.0 to 5.0 mg/ml protein or the metal-containing material), centrifuged for example at 10,000 rpm for a period of, for example, 10 to 30 minutes, suitably at 4ac. The column is then washed with the equilibration buffer until all non-binding proteins are washed, and the column is eluted, for example with Tris HCI buffer, suitably with 10 to 50 mN Tris HCI, pH 7.5 to 8.5, with a linear gradient of 0.1M to 2K NaCl. A fraction of suitable volume is collected, the wash and eluants being monitored with absorbance at 280nm. For a metal chelate complex, the pH of the buffer ia generally lowered to obtain elution. For scavenging metals, there is generally no elution by salt, although if proteins are bound along with salt elution may be necessary.

Accordingly, the present invention especially provides a process for the separation and/or purification of a polypeptide or protein, especially one having affinity for phospho-eerine, which comprises loading the poly' peptide or protein onto a chromatographic column containing a ohromatographic agent comprising phosvitin or a modified phosvitin, immobilised and coupled to a suitable 1« matrix, previously equilibrated with an equilibrating agent, and eluting the column vith a salt eolution to obtain the polypeptide or protein.

For example, for purifioation of lysozyme derived 5 from egg white, the equilibrating agent may be 66 mN KH2PO4 at a pH of 6.24, and the buffer employed for the elution may be 100 mM KH2PO4 at a pH of 6.24 containing 200 mM NaCl.

For purification of a restriction enzyme, the equilibrating agent may be, for example, 10 MM Tris-HCl having a pH of 7.5 and containing 50 nN NaCl, 5 mN MgS04 and 1 mM DTT, and the salt solution may be, for example 10 mM Tris-HCl having a pH of 7.5 and containing 1.5 M NaCl, 10 mN KCl, 100 /*g/ml BSA and 1 mM dtt.

The column operations are suitably performed at 4*C.

In the case of enzymes, activities are determined and for hormones recommended immunoassays are performed.

The following Examples illustrate the invention.

Purification of phosvitin Hen egg yolks were separated and the yolk material was obtained by puncturing the vitelline membrane and draining out the contents. The yolk contents (100 gms) were suspended in 0.11 M MgSO4, 5.5 times the voluae of the yolk material, and mixed vigorously. The mixture was kept at 4*C for 18 hours to allow precipitation. The precipitate was dispersed in 70 ml cf 0.4 M (NH<)2SO4 and the pH was adjusted to 4.0. The dispersion was nixed thoroughly and centrifuged. The supernatant was extraoS ted with 30 al of ether and this vas repeated three times. After every extraction the aqueous layer was separated. All aqueous fractions were pooled together and treated with (NH4)28O4 to give 95* saturation. The saturated aixture was allowed to stand at 4*C overnight.

The precipitated protein wee centrifuged and dissolved in water and dialysed against distilled water for 48 hours at 4’C. The water was changed every 8 hours. The dialysed protein solution was lyophilised. Yield: From 100 gas yolk material 290 mg dry protein were obtained.

The purified phosvitin was then filtered hy gel filtration to separate the a and 6 forms. For this a Sephadex 6200 column (104 x 2.5 cm) vas equilibrated with 100 mM sodium acetate buffer pH 5.0 and the dialysed protein (50 mg) were loaded onto the column. The protein in the eluate was monitored hy OD28Q. From the analysis of the phosphate content of the protein the second peak corresponded to the 0 form. The peak fractions were pooled and dialysed against distilled water. The dialysed protein solution was lyophilized. Of the 50 mg protein loaded 34 mgs were recovered in the peak - 18 fractions.

Analysis of phosvitin: Phosphate (inorganic): Phosphate estimation for the protein was done using the Ammonium Molybdate Method (Anal. Chem. [1956] 2&, 1756). The digested protein (H3SO4 * HNO3 digestion) vas subjected to analysis. The 0 form had 10.7 - 11.8% phosphorus.

Protein estimation: Lowry’s method (J. Biol. Chem. (1951], 193. 265) was used for the protein estimation.

Kleetrophoret ic analys is: Electrophoresis was done on 10% homogeneous polyacrylamide gel with Tris-glycine buffer pH 8.3. The purified protein showed a single major band. Subsequent analysis on SDS-PAOE showed that the molecular weight of the purified protein was in the range of 30,000 to 35,000. There were no major iapuritieB associated with this protein. sxftTO.i» y.

Preparation of chromatographio agent: immobilisation and coupling of phosvitin g of cyanogen bromide-activated Sepharose in freeze-dried powder form were allowed to swell in ImM HCl and washed with 1 mM HCl in a sintered glass for 15 min.

The gel vas then washed with distilled water. Phosvitin (15 mg) in the native glycosylated form prepared in - 19 Example 1 was dissolved in 25 al of o.lM NaHCOj pH 8.3 containing 0.5 M HaCl. This was mixed with the gel (10 ml) and allowed to rotate gently overnight at 4*C. The excess ligand was washed away with 0.1 K NaHCOj pH 8.3 containing 0.5 M NaCl and the remaining active groups were blocked by treatment with 1 M ethanolamine pH 9.0 for 16 hours at 4*C. The gel then was washed with three cycles of alternating pH. Bach cycle consisted of o.l M acetate buffer pH 4.0 containing 0.5 M HaCl followed by a wash with 0.1 M Tris-HCl pH 8.0 containing 0.5 M HaCl.

The coupled phosvitin-Sepharose was stored at 4 to 8*C in 100 mM Tris-HCl pH 8.0 containing 0.5 M HaCl and 0.02 % sodium azide and used for the Examples described below.

Example 3 Illustration of the binding oanaoltv of the nhosvltlnSepharoae chromatographic agent ml bed volume columns of the phosvitin-Sepharose chromatographic agent were used, which had been equilibrated with 50 mM Trie-HCl at a pH of 7.5.

To one such column, 0.25 mg of cytochrome-C (obtained froa horse heart) dissolved in the equilibrating buffer was loaded. The buffer wash showed negligible material absorbing at 410 nm (soret band of cytochrome C]. The protein was eluted froa the column with 20 mM sodium phosphate buffer pH 6.5. The recovery in the eluted sample was more than 90*.

IE 912093 Τη order to check the specificity, cytochrome-C was allowed to react with soluble phosvitin and then loaded on to the phosvitin-Sepharose. The loaded mixture did not bind to the column clearly suggesting that the binding domain on the cytochrome-C is already masked by phosvitin.

This established the efficacy of the column material, viz. phosvitin.

Example 4: Purification of lysozyme from ego White; λ phosvitin-Sepharose 4B column (5 ml bed volume) was used for this purpose. Egg white was diluted in 66 mM KH2PO4 pH 6.24 to adjust the OD28O to aPProxi*ately 10 per ml. A total of 10 ml of this diluted egg white was directly loaded onto the phosvitin-Sepharose, previously equilibrated with 66 mM KH2PO4 pH 6.24. The flow rate was maintained at 1 ml per 3 minutes using a peristaltic pump. After the loading was complete, the column was washed with 66 mM KH2PO4 pH 6.24. When the reading was below 0.05 OD, the column was eluted with 100 mM XH2fo4 pH 6.24 containing 200 mM Naci. The fractions (lml vol) were monitored for A^gg absorbance as well ae enzyme activity.

The enzyme activity was measured in 66 mM KH2PO4 pH 6.24 containing tha requisite amount of Mlcroooocus luteus cell suspension at 25 *C. One Unit is defined here as th· decrease in the optical density of o.i/min. at 450 nm under assay conditions.

The total loading in terms of enzyme unit was 4300 with a specific activity of 39.6 units per mg protein.

The salt elution gave a recovery of over 704 with a specific activity in the range of 420 and above. The three times recrystallised preparation from Sigma under identical conditions gave e specific activity of 437.

The enzyme preparation was analysed by sodium 10 dodecylsulphate polyacrylamide electrophoresis and found to contain in addition to lysozyme a small amount of ovalbumin which has a molecular weight of 45,000.

BXOTPlfi-5 Binding of ECoRI and Bam HI to Phosvitln-Senharose 15 200 units of each of these restriction enzymes obtained from Bangalore Genei Company were loaded onto separate columns of phosvitin-Sepharose 4B, which had been equilibrated with 10 mK Tris-HCl pH 7.5 containing 50 mM NaCl, S BM MgsO4 and 1 mM DTT. These enzymes were sluted with 10 mM Tris-HCl pH 7.5 containing 1.5 M NaCl, io mN kci, loo Mg/ml BSA and lmK dtt. when the salt concentration was 1.0 M, there was no enzyme elution.

The assay was based on linearization of purified PUC8 plasmid and subsequent electrophoresis of DNA on it agarose gel in Trie-Borate EDTA buffer system.

IE 912093 Exanpla β rSB binding and elution Phosvitin-Sepharose 4B column (0.25 ml bed volume) wae used. Tha column was equilibrated with 10 mM Tris HCI pH 7.75. The column was loaded with human Follicle Stimulating Hormone (corresponding to 7 units/1). The column was washed with the equilibration buffer and 8 fractions of 0.25 ml were collected. The column was then eluted with lOmM Tris HCI pH 7.75 containing im NaCl and 8 fractions of 0.25 ml were collected. Tha fractions were subjected to Delfia ®assay. FSH was quantitatively recovered in the third fraction of the eluting buffer.

Example 7 Affinity of Phosvitln-Senharose to Adrenocorticotropic Hormone /ACTH) Phosvltin-sepharose (0.25 ml bed volume) was equilibrated with 10 mH Tris HCI pH 7.7. 200μ1 of ACTH (human) l mg/ml was loaded on the column and the column washed with the same buffer. The column was first eluted with 10 mN Tris HCI pH 7.7 containing 1 K NaCl. Ths wash and salt eluates were assayed for ACTH by radioimmunoassay. It was observed that ACTH is bound to tha column and is eluted by 1.0 N NaCl.

Example 8 Affinity of Phoavltln-Bepherose to Parathyroid Hormone (PTH 44-68) Phoevitin-Sepharoee (0.25 ml bed volume) was 5 equilibrated with 10 >H Tris HCl pH 7.7. 200 μΐ of parathyroid hormone (PTH) (44-68) 872 pmole/1 vae loaded. The column was first eluted with 10 mM Tris HCl pH 7.7 containing 1 H NaCl and the seoond elution was done with 10 mM Tris HCl pH 7.7 containing 1.5 m NaCl.

Assay for the wash and salt eluates vas done by radioimmunoassay. It was observed that no elution was obtained with 1 K NaCl, and 1.5 M NaCl vas required for eluting PTH from the phoavitin-Sepharose column, indicating strong binding.

It is clear from Examples 7 and 8 that the phosvitin-Sepharose also binds to hACTB and hPTH from serum samples.

EXAMPLES WITH MQDiriED.EflQgviTIH Bxsuplfi-a Modification of Phosvitin Phosvitin was Modified by proteolysis of the native protein with trypsin aa follows· Zrypsixiiaation: Phoevitin (purified on the Mono Q column on the FPLC system) vae taken up in 20 mM Tris HCl pH 7.5 at a concentration of 20 mg/ml and was treated with trypsin (in a phosvitin:trypsin ratio [w/wj of 100:1). The mixture was incubated at 37°C for 1 hour. On SDS-PAGE the native protein showed 3 major bands around 28-32kDa. The trypeinised phosvitin showed a major band at 26 kDa and two others corresponding to molecular weights of around 8000-14000.

Purification of trypeinised phoevitin: The trypeinised phoevitin vas subjected to Cu(ll) imino di-acetio acid, metal chelation chromatography.

For this purpose chelating Sepharose fast flow column (1 ml, Pharmacia) was equilibrated with a solution of 50 mM copper sulphate till the entire column was coloured. The column was then equilibrated with 20 mM sodium phosphate buffer pH 7.5 containing 0.5 M NaCl and then it was loaded with 200 μΐ (2 mg) protein solution. The column was developed with a pH gradient generated using 20 mM sodium phosphate buffer pH 7.5 containing 0.5 M NaCl (buffer A) and 200 mM sodium phosphate buffer pH 3.5 containing 0.5 M NaCl (buffer B). All operations were performed on the FPLC system (Pharmacia) at 2l*C. Some A280 absorbing material (i.e. that absorbing at 280 nm) (peak Pl) was eluted in the buffer A wash and later, at the end of the gradient, the pH was continued to be maintained at 3.5 when another protein (peak P2) MSB - 25 confirmed that peak P2 represents a truncated version of phosvitin which corresponds to a molecular weight of 26000.

Analytical Results: B Although tho molooular weight of truncated phnsvIMn was less by about 4000-5000, compared to the native protein, their phosphate and carbohydrate content remained essentially similar.

Example ltt Immobilisation of truncated phosvitin The P2 fraction was dialysed against 5 mN EDTA and then against Mill! Q water. The dialysed protein was lyophilized, and covalently attached to CNBr-activated Sepharose (protocol of coupling was the same as described for phoevitin-Sepharose). 1 ml of coupled matrix typically contains 6 mg protein.

The coupled matrix was designated as P2-Sepharose and was tested for its binding characteristics as described below.

Example 11 Affinity of Cytochroae-C to P2-Sepharose - - ' ' ----------,.,,-¼¼ was nearly zero, and than eluted with 20 mM Trie HCl pH 7.5 containing 1 M Nacl. The aalt-eluted fractions showed the soret band at 4io na. The sodium dithionite reduction which resulted in the appearance of a, Β I / bands confirmed the presence of cytochrome-C.

The binding characteristics of cytochrome-C to P2Sepharoee were very similar to those of phosvitinSepharose matrix.

Example 12 Affinity of Lvbozvbc to P2-Sepharose The 0.5 ml bed volume of P2-Sepherose column was equilibrated with 20 mM Tris HCl pH 7.5. The column was loaded with 500 μΐ of 14 mg/ml lysozyme (Sigma) . The matrix was washed with the equilibration buffer till the A280 was zero, and then eluted with 20 mM Tris HCl pH 7.5 containing IX NaCl. virtually no A280 absorbing material appeared either in the breakthough or in the wash fractions. There was essentially total recovery (>85%) of lysozyme in the salt-eluted fractions. The presence of lysozyme in the eluate was confirmed by subjecting the fractions to enzyme assay. P2-Sepharose has a capacity to bind to 13-14 mg of lysozyme per ml of gel bed volume, whereas the equivalent capacity for phosvitin-Sepharose is 5-6 mg/ml.

The column was used for purification of lysozyme from egg white. The crude egg white preparation was - 27 diluted to 10 OD (A260)/Bl with 20 mM Tri· HCl pH 7.5.

The P2-Sepharose column (l ml) was loaded with 50 OD of crude protein. The breakthrough protein did not show any appreciable lysozyme activity, whereas the 20»M Tris HCl pH 7.5 containing 1.0 H NaCl eluates showed very high specific activity.

In one experiment 7.5 mg of lysozyme could be purified with specific activity of 425 u/mg protein (Unit definition: 1 unit of lysozyme causes A450 change of io O.l/min at 25°c at pH 6.24).

Example 13 Purification of EcoRI on P2-Sepharose EcoRI was purified from the extract of strain RY13 of IL· coll. The extract of £,. coll (RY13) was prepared according to the following protocol: The strain was grown on L-broth pH 7.0 (tryptone 10g/l; NaCl 10 g/1: Glucose 5 g/1 and Yeast extract 5 g/1), till the 00660 was 1.0 to 1.1. The cells ware harvested by centrifugation at 10000 rpm for 10 min and washed in TGM (20 mM Tris HCl pH 7.5 containing 2mN EDTA and 1 mM A-mecaptoethanol) . The cells were taken up in TEM and lysed by sonication for 10 min using Sonics and Material; Vibra Cell Miorotip. The lysed extract was centrifuged and the supernatant waa treated with 5% (final concentration) streptomycin sulphate at 4*C for 45 min. The solution was centrifuged at 15000 rpm for 15 •Ε 912093 - 28 ηίη. and the supernatant was dialysed against TEN for 24 hours. The dialysed extract had typically 8.0 mg/ml protein. ml P2-Sepharose was equilibrated vith TEN buffer, and 2 ml of the extract was loaded on the column. The column was washed with TEN till the A280 was zero and then eluted with TEN containing 1.5 N NaCl. To the peak fractions, BSA (bovine serum albumin) was added to a final concentration of 100 pg/ml and the peak fractions dialysed against ten overnight and assayed for EcoRI activity.

Ve have used lambda-DNA digestion assay to detect and quantitate the EcoRI activity. We can purify typically 5500 units of EcoRI using 1 ml bed volume of IS P2-Sepharose.

Claims (26)

1. λ process for the separation and/or purification of a polypeptide or a protein or for the removal of metal ions from biological material wherein there is used a 5 chromatographic agent which comprises phosvitin or a modified phosvitin immobilised and aoupled to a suitable matrix.

2. λ process for the separation and/or purification of a polypeptide or protein having affinity for phospho10 serine, which comprises loading the polypeptide or protein onto a chromatographic column containing a phosvitin chromatographic agent which comprises phosvitin or a modified phosvitin immobilised and coupled to a suitable matrix, previously equilibrated with an equi15 librating agent, and eluting the column with a salt solution to obtain the polypeptide or protein.

3. A process as claimed in claim 1 or claim 2, wherein the matrix is a Sepharose gel.

4. λ process as claimed in claim 3, wherein the 20 Sepharose gel is a CNBr-activeted Sepharose gel.

5. A process as claimed in any one of claims 1 to 4, wherein the phosvitin is modified by proteolysis with trypsin.

6. A process as claimed in any one of claims 1 to 25 5, wherein the phosvitin or modified phosvitin is in the form of a metal chelate complex.

7. A process as claimed in claim 6, wherein the - 30 metal la Fa 2 *, Fe 3 *, zn**, Cu**, Mn 2 * or Ca**.

8. A process as claimed in claim 1 or claim 2, wherein the phosvitin or modified phosvitin is substantially as described in Example 2 or Example 10 herein. 5

9. A process as claimed in anyone of claims 1 to 8, wherein the protein is lysozyme, cytochrome-C or a restriction enzyme.

10. A process as claimed in any one of claims 1 to 8, wherein the protein/polypeptide is any one of those 10 mentioned in Table 1 herein.

11. A prooese as claimed in claim 6 or claim 7, wherein the chromatographic agent is used for the separation and/or purification of a metal-dependent enzyme or another protein. 15

12. A process as claimed in any one of claims 1 and 3 to 8, which comprises the removal of excess iron from physiological fluid.

13. A process as claimed in claim 1, carried out substantially as described in any one of Examples 3 to 8 20 and 11 to 13 herein.

14. A separated and/or purified polypeptide or protein, or biological material freed from metal ions, whenever obtained by a process ae claimed in any one of claims 1 to 13. 25 15. Use of phosvitin or a modified phosvitin for the preparation of a chromatographic agent for the separation and/or purification of a polypeptide or protein or for the removal of metal ions from biological material. 16. Phosvitin or a modified phosvitin, immobilised and coupled to a suitable matrix, for use in tha 5 separation and/or purification of a polypeptide or protein. 17. A modified phosvitin, for use in the separation and/or purification of a polypeptide or protein or in the removal of metal ions froa biological or other material. io 18. Phosvitin or a modified phosvitin, in the form of a metal chelate complex, for use in the purification of a polypeptide or protein. 19. A modified phosvitin as claimed in claim 17 or claim 18, wherein the modification is by proteolysis with

15. Trypsin.

16. 20. A modified phosvitin as claimed in claim 17, substantially as described in Example 9 herein.

17. 21. A chromatographic agent which comprises a modified phosvitin, immobilised and ooupled to a suitable 20 matrix.

18. 22. A chromographia agent as claimed in claim 21, wherein the modification is by proteolysis with trypsin.

19. 23. A chromatographic agent as claimed in claim 21, substantially as described in Example 10 herein.

20. 25 24. λ chromatographic agent which comprises phosvitin in the form of a metal chelate complex, immobilised and coupled to a suitable matrix. 25. λ process for the preparation of a chromatographic agent ae claimed in olaim 21 or claim 24, vhich comprises mixing phosvitin or a modified phosvitin with the matrix in the presence of a buffer, washing away the 5 excels ligand of the phosvitin end blocking the remaining active groups of the matrix by treating the mixture with an amine to produce coupled phosvitin-matrix and washing the resulting product.

21. 26. A process as claimed in claim 25, wherein the 10 buffer is such that the pH of the mixture ie in the range of from 8.0 to 8.3.

22. 27. A process as claimed in claim 25 or claim 26, wherein blocking is carried out with a primary amine.

23. 28. λ process as claimed in any one of claims 25 to 15 27, wherein the washing of the resulting product comprises two or more cycles of alternating pH.

24. 29. λ process as claimed in any one of claims 25 to 28, vherein the phosvitin ia mixed with the matrix in an amount of 6 to 10 mg phosvitin per ml of swollen matrix. 20

25. 30. A process as claimed in olaim 25, carried out substantially as described in Example io herein.

26. 31. A chromatographic agent as claimed in claim 21 or claim 24, whenever prepared by a process as claimed in any one of claims 25 to 30. 25 32. A process for the removal of peroxide from a solvent, wherein there is used phosvitin or modified phosvitin in the form of an iron chelate complex. - 33 immobilised and coupled to a suitable matrix.