NO175782B - Process for the preparation and purification of amphiphilic proteins in aggregate form produced in cells and their use - Google Patents

Abstract

Proteins produced in cells collect together to form particulate aggregates. The invention relates to the reversible conversion of said aggregated proteins into morphological forms I, II and III which differ from one another, depending on the presence or absence of a non-denaturing detergent. The invention also relates to a process for the isolation and purification of said aggregated forms of proteins produced in cells.

Description

The invention relates to a method for isolating and purifying soluble, amphiphilic proteins produced in cells and their use.

All physiologically active cells produce proteins that they need to maintain their vital functions. The synthesized proteins have different tasks. Some act as enzymes, i.e. they catalyze the metabolic reaction in the cell. Others serve to coordinate the life course of the organism to which the cell belongs, and still others form the cell structure. Proteins produced in these cells can nevertheless have antigenic effects on other organisms, i.e. they induce immune reactions in these organisms. Viruses are also living beings such as e.g. in its capsule contains proteins that can act as antigens.

The instructions for protein synthesis are found in the cells' genes, which can also be considered the functional units of the genetic material. It is today a well-controlled technique to change the genetic equipment of a cell, e.g. a bacterium, transferring a gene e.g. from an animal or human cell to another cell, preferably a bacterial cell. The bacterium then also synthesizes the protein that the foreign, inserted gene codes for. One of the most famous examples is insulin produced in the human pancreas, whose genetic information was transferred to the bacterium Escherichia coli.

Both the natural proteins and the proteins produced by genetic manipulation of cells are either carried out of the cell through an overflow or accumulate in the cell and are then isolated by known methods and possibly dissolved after the cell has been broken up. For these methods, as a rule, after certain fractionation and purification pretreatment steps, detergents are used, which must be coordinated very specifically for the isolation and purification of the individual proteins. It is now as before a problem according to the state of the art to find the right detergents for isolating and purifying the desired proteins. It was therefore an aim of the invention to provide methods by means of which proteins produced in physiologically active cells can be isolated and purified, and thereby maintain or improve the proteins' biological activity.

This purpose was achieved by obtaining proteins that exist in a first, approximately spherical, possibly electron-optically visible, aggregated, electrophoretically non-mobile form I in the absence of detergents, in a second, loose, possibly electron-optically visible, aggregated, electrophoretically not -mobile form II in the presence of a first, non-denaturing detergent, and in an electron-optically non-visible, electrophoretic mobile form III in the presence of a second, non-denaturing detergent, and that said forms I, II and III are reversible transferable in each other depending on the presence or absence of the aforementioned detergents.

In isolation procedures for proteins, it has surprisingly been shown that amphiphilic proteins, when non-denaturing detergents are used, are not present in their monomeric form, but in aggregated forms, which quite surprisingly exhibit advantageous properties. In the absence of a non-denaturing detergent, the aggregated forms exhibit a compact structure which will be referred to here as aggregated form I. In this form, the particle is best suited for removing contaminants that settle on the surface of these particles. The compact form of the particles also ensures a certain protection against denaturation of the active centers, be it enzymatically active centers or epitopes that cause immune reactions. Nevertheless, the compact particles are also bioactive in the aggregated form I, but not electrophoretically mobile.

In the presence of a non-denaturing detergent, the compact globular form I of the aggregated protein allows itself to be transferred into a more or less airy form, which during an isolation and purification process exhibits the advantage that contaminants which through the transition from form I to form II become more easily accessible, can be removed. In form II of the aggregated proteins, the proteins' biological activity is surprisingly enhanced. It turns out, for example, that the immunogenicity of the isolated protein increases, when the biological property of the isolated proteins lies in the proteins' immunizing antigenicity. The biologically active proteins or possibly also parts of these proteins, which exhibit a biological activity, are thus active, especially in the expanded form, and are suitable in this cloud-like form for the reasons stated above particularly well for purifying the protein for components that are contained in the aggregated form.

The protein in the aggregated form II is likewise electrophoretically non-mobile.

What is particularly surprising about the particulate aggregated proteins that can be produced according to the invention is the fact that the aggregated forms I and II are reversibly transferable into each other, depending on the absence or presence of a first non-denaturing detergent. With the described particulate aggregated proteins, a very suitable complex is thus made available, which due to the reversibility of the transfer of the different forms I and II of these aggregates into each other in consideration of a simpler purification process, an improved storage possibility as well as an increased activity of the biological activity of the isolated proteins exhibits enormous advantages.

When another non-denaturing detergent is added in a controlled manner, the protein can be transferred from the aggregated form II to the monomeric form III of the proteins, while this condition is also completely unexpectedly reversible when the other, non-denaturing detergent is removed. Depending on the absence or presence respectively of the non-denaturing detergents, proteins to be isolated from physiologically active cells can be controlled and manipulably reversibly transferred into any desired form that is optimal for the intended method step resp. method of application.

For the storage or a dosed, possibly desired, reduced biological activity of the isolated proteins, an aggregated form I with an approximately spherical structure is preferred. The spherical shape further protects possible sensitive active centers in the cleaning step against possible denaturing treatment steps.

Depending on the duration of the treatment and the concentration of a non-denaturing detergent, the aggregated form II will exhibit an approximately cloud-like (airy) structure. Depending on the degree of unfolding, the protein can be subjected to further purification steps for the removal of such impurities which may originate from e.g. the host cells and vector systems used in the production of the proteins. The biological activity of the proteins is increased.

Both the globular aggregated form I and the cloud-like aggregated form II can be made electron-optically visible when the single protein produced exhibits a molecular weight of at least 10,000 Daltons. When, however, e.g. by genetic manipulation, only certain antigen determinants are expressed that make up proteins with few amino acids, the aggregated, more protein-rich forms I and II can also lie below what is electron-optically visible.

Preferably, the aggregated, electrophoretically non-mobile proteins in forms I and II are present in a purity of 80%, preferably 98%, i.e. that the aggregated forms I and II contain the desired proteins in relatively homogeneous accumulation.

The aggregation of the proteins inside the cells preferably takes place when the proteins are expression products of genetically manipulated cells and especially when the products are overproduced in these cells using known methods. The overproduction can take place by either several vectors containing the gene expressing the desired protein being present, or by particularly strong promoters being linked in front of the corresponding genes. When the expressed proteins are soluble in the cell, these agglomerate into the spherical particles described.

The proteins expressed by the genetically manipulated host cells preferably exhibit an antigenic function. These proteins are preferred projects for the genetically engineered expression of proteins when live vaccines are to be provided or a correspondingly active immunization via the antigens is intended. This particularly concerns coat proteins of pathogenic viruses, be they human pathogenic or animal pathogenic viruses.

Preferably, the isolated proteins are glycoproteins, e.g. the glycoprotein GP 160 of HIV or a surface antigen of the hepatitis B virus. The genes for expression of the respective proteins can e.g. is expressed through a Vaccinia virus vector in a mammalian cell system. The procedure for inserting specific genes into Vaccinia virus vectors is described by Mackett et al. (J. Virol, 49, pp. 857-846, 1984). In a manner known per se, the foreign gene is first inserted into a plasmid vector downstream of a Vaccinia transcriptional control element (7.5 K promoter). This chimeric gene in the recombinant plasmid is flanked by Vaccinia sequences that encode the viral thymidine kinase (TK) gene. The plasmid is then introduced into cells previously infected with a wild-type Vaccinia virus (strain WR). The recombination then takes place in the TK region, which is homologous to both the viral DNA and the plasmid and allows insertion of the chimeric gene into the genome of the Vaccinia virus. The recombinant virus obtained in this way exhibits the phenotype TK~. It is thus no longer able to produce thymidine kinase and grows in selective medium containing 5-bromodeoxyuridine. In this way, recombinant Vaccinia viruses can be produced that each carry genes that produce the proteins, possibly in overproduction, which then agglomerate in the described mammalian cells into the described aggregated forms that contain a number of the individual proteins. Similar expression systems are known for other antigenic proteins. E.g. certain Vaccinia virus recombinants have been constructed that code for the surface antigens of hepatitis B virus. For this purpose, the hepatitis B virus surface antigens were expressed under the regulatory mechanisms of the Vaccinia virus, using a number of Vaccinia-specific promoters with the aim of increasing the expression level of the foreign genes. Vaccinia virus recombinants have also been produced which express more than one hepatitis B virus gene.

In general, at this point, with regard to the production of recombinant expression systems for the genetic engineering production of desired proteins, reference should be made to Winnacker, E.L. (Gene und Klone, eine Einfuhrung in die Gentechnologie, VCH-Ver-lagsgesellschaft mbH, Weinheim, 1984).

The biological function of the proteins which are expressed in the aforementioned cells, and which may exist in aggregated forms which may contain many proteins, may preferably be an enzymatic function. Particularly in the aggregated form II, an enhanced enzymatic function is observed, which may be related to the fact that the enzymatically active centers in the airy, aggregated form II are free, but that there is nevertheless a spatial connection between all enzymatically active proteins, so that the probability of coincidence between the products to be converted by the enzymatic activity of the proteins with the enzymatically active regions is increased.

The function of the above-mentioned protein can be elucidated by the enzymatic protein for blood coagulation factor VIII. The genetic engineering of F VIII has been described several times in the state of the art. The proteins agglomerated in particle form can preferably be bound to a further substance, in particular a high molecular weight protein, by conjugation, whereby an improvement with regard to stability, activity, in-vivo recovery and biological half-life of the protein is achieved. An example of such a conjugation between desired proteins and additional high molecular weight substances is the production of blood coagulation factor VIII with that from Willebrand's factor.

The proteins aggregated in particle form are preferably present

in eukaryotic cells, especially animal cells or human cells.

In this connection, it is particularly preferred to express the desired proteins in primary cell cultures or Vero cells obtained from cell lines, CHO cells or primary chicken embryoblast cells. The aforementioned host cells are preferred as expression systems, since here already the modifications that take place after the translation are present in the way that is desirable in the final product, e.g. the glycosylation of the expressed proteins. These glycosylated proteins then clump together into particle form which can assume the described spherical form I resp. the airy form II after treatment with a first non-denaturing detergent and after treatment with a second non-denaturing detergent can be transferred into a third aggregated form III.

Vero cells are particularly preferred for the production of glycoprotein 160 of HIV, as described above. This cell system was chosen, as it has a relatively fast growth rate and is particularly well adapted to the production of human vaccines. The infection of the Vero cells with a recombinant Vaccinia virus, which contains a gene expressing the desired protein to be isolated according to the method, results in normal synthesis of the desired protein, its glycosylation as well as any further processing steps. By using a double-infection system with two recombinants, the amount of the desired protein can be increased considerably. This system contains a recombinant expressing the polymerase of phage T7 under the control of the Vaccinia p7.5 promoter. This recombinant is co-infected with a recombinant containing the T7 promoter and the glycoprotein-160 gene of HIV. The T7 promoter is a very strong promoter and ensures a high expression level, e.g. an approx. 10-fold amount of the desired protein.

A general overview of the numerous possibilities for using specific vectors for the recombination of the desired genes and propagating these vectors in desired cells and producing the proteins expressed by the foreign genes can be found, for example, in Genetic Engineering, 3, Academic Press, edited by Robert Williamson, ( 1982) or in Spektrum der Wissenschaft, Industrielle Mikrobiologie (1985).

The non-denaturing detergents, the absence or presence of which significantly affects the occurrence of the proteins isolated from the physiologically active cells in mutually different aggregated forms or in monomeric form, is preferably a specific ionic, non-ionic or zwitterionic detergent. It is within protein chemistry or with protein purification methods, a known problem is that the choice of detergent for the recovery of the desired protein in, on the one hand, a very pure form and, on the other hand, in a biologically active state, each time represents a challenge. It has now surprisingly been shown that the above-mentioned requirements for a protein purification method can be fully satisfied by the use of non-denaturing detergents of a specific ionic, non-ionic or zwitterionic nature, at the same time that the unexpected change in configuration of the aggregated proteins present in these protein-producing cells, to an astonishing degree, optimally meet the aforementioned requirements. As previously mentioned, particularly in the aggregated form II, that is to say the airy or fluff-like configuration, the biological activity inherent in the individual protein, whether this is of an enzymatic or antigenic nature, is clearly enhanced.

A preferred non-ionic detergent is octyl glycoside or one of the derivatives of this detergent, preferably in concentrations of 0.25-5%, especially 1%.

Equally preferred is the use of a detergent of the salt of bile acid as well as one of its derivatives, preferably the detergent deoxycholate (DOC), which is particularly suitable in connection with the isolation and purification of the globular configuration of many proteins of glycoprotein 160 of HIV, both when this is cleaned using the above-mentioned detergent in concentrations of 0.25-5%, preferably 1%, and also when the deoxycholate is used when using GP 160, e.g. in

compound with aluminum hydroxide.

The non-denaturing detergent that is preferred to be used for the transfer of the aggregated proteins from form II to the monomeric form III is a zwitterionic detergent from the ZWITTERGENT series, likewise in concentrations of 0.25-5%, preferably 1%. ZWITTERGENT<R->detergents are synthetic zwitterionic detergents that have become known as sulfobetaines and have the following general structural formula:

The proteins in the aggregated form I are, as previously stated, particularly suitable for the stable storage of the proteins. However, they can also be used for the desired biochemical reactions or reactions, as the aggregated proteins in the aggregated form I also exhibit biological activity.

The use of the proteins in the aggregated form II preferably takes place to enhance the protein's biological function.

Any aggregated form of the proteins is suitable for a therapeutic, prophylactic or diagnostic application, both in the described globular, tuft-like or monomeric forms and in the above-described conjugated forms with additional high molecular weight proteins.

An application of the aggregated protein lies in obtaining

substances for qualitative detection and quantitative determination of such substances in body fluids.

The present invention relates to a method for the isolation and purification of a number of particles consisting of proteins, which, depending on the presence or absence of a non-denaturing detergent, exist in morphologically different configurations, between which they can be transferred reversibly, which are produced in cells, preferably genetically manipulated cells, in which the surface contaminants of the proteins present in the first aggregated form I are removed in a first purification step, the proteins are then, by using a non-denaturing detergent, transferred into another aggregated form II, those in this aggregated form II available contaminants are removed in another purification step, the proteins by adding another, non-denaturing detergent are made transferable in a monomeric form III and the proteins by removal of the non-denaturing detergents again become reversibly transferable in the aggregated forms I and II .

The enormous advantages of this method are essentially discussed above and lie in the reversible transferability of the different aggregated forms of the protein to each other.

As a result of the compact arrangement of the proteins, the approximately spherical form I allows, for example, an easier initial cleaning of, for example, media constituents from the surface of the approximately spherical protein agglomerates. When the second cleaning step of contaminants which are particularly easily accessible in the aggregated form I as a result of expansions has then been completed, the aggregated form I can again be produced reversibly by extracting the non-denaturing detergent. In this form, they are e.g. active centers of the proteins particularly well protected in the interior of the spherical protein agglomerates, so that a particularly good storage of the proteins in the aggregated form I is possible. As already mentioned, the aggregated form II has, in addition to the advantages with regard to easier purification of cellular components of the host cell, in which the proteins are expressed, resp. of the vectors, which recombinantly contain the gene for the isolated proteins, the further advantage that the biological activity of the proteins can be significantly enhanced as a result of the spatial proximity of many active centers in the special aggregated state of the protein.

Depending on the experimental arrangement, cells or cell membranes are centrifuged and extracted with a buffer solution containing a non-denaturing detergent and a protease-inhibiting system, or (when the aforementioned high-molecular-weight substance is released from the cells in the overflow) the overflow mixed with the non-denaturing detergent and the protease-inhibiting system , as the non-denaturing detergent, e.g. deoxycholate (DOC), present in concentrations of 0.25-5%, preferably 1%.

When it comes to purification and isolation, the solutions thus obtained are concentrated and separated from non-hydrocarbon-containing contaminants by a glycoprotein with the help of lectins which are linked to a solid matrix. In this step, the non-denaturing detergent used has, in addition to the property of transferring the substance in form II, the ability to prevent non-specific adsorption. The elution is carried out with glycosides.

A further essential purification step can be achieved by special binding of the pre-purified high molecular weight substance to an immunoadsorption column. After sufficient washing with a buffer, elution is carried out with a chaotropic agent, e.g. urea or KSCN (2-8 M, preferably 3M). With direct subsequent dialysis against a buffer, the substance is transferred in particle form I. If necessary, the high molecular weight substance is transferred in monomeric form II with the help of a specific zwitterionic detergent and purified by ion exchange chromatography. By dialysis against a detergent-free buffer, the aggregated forms II or I are again produced from the monomeric form III. The different aggregated forms in the described cell-produced proteins can be made electron microscopically visible.

The invention shall be illustrated in detail by means of the following

figures and examples.

Fig. 1 shows an electron microscopic photograph of glycoprotein 160 in Tris buffer at 278,640 times magnification.

The proteins exist in the compact, aggregated globular form I.

Fig. 2 shows an electron microscopic photograph of the glycoprotein 160 from HIV in a buffer containing 1% DOC.

The proteins exist in the airy, tuft-like aggregated form II.

Example 1

Isolation and purification of glycoprotein 160 from HIV.

The isolation and purification of glycoprotein 160 from HIV took place according to the following scheme: 1. Genetically manipulated Vero cells, which produce glycoprotein 160 of HIV, were centrifuged at a desired density. 2. The pellet recovered by centrifugation was resuspended in TBS pH 7.4 plus 1 mM CuSO 4 + 0.5 mM ZnCl 2 . 3. The resuspended pellet was subjected to an extraction with 50 mM Tris-HCl pH 8.3, 1% DOC and 1 mM CuSO 4 + 0.5 mM ZnCl 2 . The suspension was cleared by centrifugation for 30 min. at 25°C. 4. The supernatant liquid was subjected to lentil/lectin chromatography, where the desired proteins were adsorbed on a column and washed with DOC buffer. The proteins were eluted with 5% methylglycoside in a wash buffer. 5. The eluate from the lectin column was diluted in a Tween buffer. This was followed by adsorption on an immunoaffinity chromatography column, which was washed with TBS-Tween and TBS. A DNA and RNA treatment followed. There was eluted with 3M KSCN and dialyzed against detergent-free buffers. 6. The dialyzed eluate was adjusted with 1% Zwittergent and 5% betaine and then adsorbed on a mono-Q matrix as an ion exchanger and then eluted with a KSCN gradient and dialyzed against a buffer. 7. Another lentil/lectin chromatography with adsorption and washing with detergent-free buffer was performed. The elution was performed with 5% methylglycoside, followed by a dialysis against TBS. At this method step, a concentration of the protein took place.

Example 2

With glycoproteins (GP) 160 of HIV purified by the method described in example 1, an efficacy test was carried out which shows the immunogenic effect of glycoprotein 160 in compact, globular form I as well as in airy, fluffy form II.

For each vaccine, 5 dilutions were prepared with respectively 10 µg, 2.5 µg, 0.625 µg, 0.158 µg and 0.04 µg GP 160 per ml. 10 BALBc mice were subcutaneously immunized with each dilution. For each vaccine type, 50 animals were therefore needed. In the present experiment, four different adsorption methods were tested. 1. GP 160 + 0.2% A1(0H)3 2. [GP 160 + 0.25% DOC] + 0.2% A1(0H)3 3. [GP 160 + 0.25% DOC] + [ 0.2% A1(0H)3 + 0.25% DOC]

4. [GP 160 + 0.25% DOC] + [0.2% Al(OH)3) washed]

All animals were immunized subcutaneously with 1 ml, and after six weeks each animal was bled. The serum from each animal was examined with anti-HIV ELISA (SORIN) for the presence of antibodies against GP 160, the effective dose 50 of GP 160 for each vaccine type being calculated using the reacting animals by the known Spearman-Kaerber method.

Results

1. I-V 10 ug GP 160/ml + 0.2% Al(OH)3 s.c.

2. VI-X 10 /ig GP 160/ml + DOC + 0.2% Al (OH) 3 s.c.

ED50: 3.3 /ug/ml GP 160

3. XI-XV 10 /zg GP 160/ml + DOC + DOC-Al(OH)3 s.c.

ED50: 1.09 ng/ial GP 160

4. XVI-XX 10 ixg GP 160/ml + DOC + Al(OH)3 washed s.c.

The result of the experiment with the effect of glycoprotein 160 of HIV shows the different immunogenicity towards glycoprotein 160 depending on the aggregated form. The ED50 value, which denotes the quantity of the substance used at which 50% of the vaccinated animals seroconvert, is reduced considerably when the detergent DOC is present. In the presence of the detergent DOC, the glycoprotein 160 is present in the aggregated form II, where the antigenic epitope on the one hand is free and on the other hand hangs together in space, so that an enhanced immune response is obtained.

The invention is illustrated with respect to glycoprotein 160 of HIV as an example. However, it is by no means limited to this protein.

Claims (8)

1. Method for isolating and purifying glycoprotein GP 160 of HIV produced in cells, characterized in that the contaminants that are on the surface are removed in a first cleaning step from GP 160 which is in a first aggregated form I, that GP 160 using a non-denaturing detergent selected from certain non-ionic or zwitterionic detergents is then transferred to a second aggregated form II, and that in this aggregated form II available contaminants are removed in a second cleaning step and possibly that by adding a second, non-denaturing detergent, GP 160 is transferred to a monomeric form III, and that this by removal of the non-denaturing detergent is again reversibly transferable to the aggregated form I or II. 2. Method as stated in claim 1, characterized in that octylglucoside or a derivative thereof is used as the first, non-ionic detergent, preferably in a concentration of 0.25-5%, especially 1%, and that as the other zwitterionic detergent is used one from the ZWITTERGENT range, preferably in a concentration of 0.25-5%, especially 1%. 3. Method as stated in claim 1, characterized in that a salt of bile acid or a derivative thereof, preferably deoxycholate (DOC), preferably in a concentration of 0.25-5%, especially 1%, is used as detergent. 4. Method as stated in claim 1, characterized in that the reversible transfer of the aggregated forms I, II and III is carried out several times for purification purposes. 5. Method as stated in claim 1, characterized in that GP 160 is an expression product of genetically engineered cells, especially those that overproduce the proteins. 6. Method as stated in one of claims 1 or 5, characterized in that the cells are eukaryotic, preferably animal cells or human cells. 7. Method as stated in claim 6, characterized in that the cells are obtained from primary cell cultures or from cell lines, in particular Vero cells, CHO cells or primary chicken embryo fibroblast cells. 8. Use of glycoprotein GP 160 from HIV in form II to obtain substances for qualitative detection or quantitative determination in vitro of the said substances in body fluids.