WO2013026445A1

WO2013026445A1 - Method for quickly isolating or purifying recombinant proteins using a (non-toxic) cholera toxin domain

Info

Publication number: WO2013026445A1
Application number: PCT/DE2012/000877
Authority: WO
Inventors: Holger PRIETZSCH; Juliane KLÜSS; Alain Steinmann; Udo Meyer; Nadine KOLP
Original assignee: Bioserv Analytik Und Medizinprodukte Gmbh
Priority date: 2011-08-24
Filing date: 2012-08-24
Publication date: 2013-02-28
Also published as: DE102011111688A1

Abstract

The invention relates to a method for quickly, efficiently, and easily purifying proteins of protein or substance mixtures. The binding of a toxin (such as a cholera toxin) to the corresponding receptor (such as the GM1 receptor) is used, the binding being carried out not only in a very specific manner but also with high affinity. Said binding properties (affinity, specificity) are used in the invention in that a fusion protein is expressed which has a toxin-binding domain. The fusion protein can be used as a whole or only partly (without the toxin-binding domain) after being cleaved by a protease at a defined point. The aim of the invention consists in providing a quick and highly economical purification for proteins. The method can be used in medicine, veterinary medicine, science, research, and different branches of industry.

Description

Process for the rapid isolation or purification of recombinant proteins with a (non-toxic) cholera toxin domain

The invention relates to a process for the rapid isolation or purification of recombinant proteins with a (non-toxic) cholera toxin domain. Fields of application of the invention are medicine, veterinary medicine, science and research as well as various branches of industry

Cholera is a severe, bacterial infectious disease predominantly of the small intestine, caused by the exotoxin (also called cholera toxin) of the pathogen Vibrio cholerae. The infection usually takes place via contaminated drinking water or infected food in which the bacteria may be present. The result of infection is extreme diarrhea and severe vomiting, resulting in very rapid dehydration with electrolyte loss. Although most infections (about 85%) have no symptoms, between 100,000 and 120,000 people still die every year from this disease, especially in the developing world (WHO, 2011). Due to the dangerous nature of the pathogen, it was investigated intensively at an early stage. As a result, cholera toxin was one of the first toxins to be described in detail and functionally and in terms of its molecular structure. Bacterial toxins are for microorganisms biological instruments to prevail in a hostile environment. Typically, the classical toxins are released by the microorganisms into the environment to act there in or on the cells regardless of the presence of bacteria. For this, the microorganisms have developed a surprising number of techniques to attack the host organism. These properties of protein toxins are often exploited to exploit them as pharmacological tools in the study of cell biology of more sophisticated cells. Among these toxins that act on the cell surface are those that form a formation with a pore. Other toxins only work in the cytoplasm of cells and thus allow the study of the mechanisms of how they can penetrate into the cells. These protein toxins are not only highly specific, but also very effective and extremely efficient. Even though these toxins are very large, many can still invade the cell without significantly damaging the cell membrane. This high specificity and efficiency of the toxins is very important for use as

CONFIRMATION COPY scientific tool. Many of these toxins can distinguish extremely precisely between (evolutionarily) related and structurally very similar target structures of the host organisms. A bacterial toxin from this group is cholera toxin, which was one of the first toxins to be analyzed in detail and scientifically extensively described. As part of the global research, the complex immunological properties were investigated (protective immunogen, effective mucosal adjuvant, immunomodulator). In particular, non-toxic subunit B (CTB) is used in a mucosal vaccine for the induction of effective intestinal immunity to cholera toxin so as to protect against cholera disease. Frequently, additional cross-immunity to enterotoxic Escherichia coli is also observed (Clemens et al., 1988, Peltola et al., 1991, Scerpella et al., 1995). The toxin itself consists of 2 subunits, the cholera toxin A (CTA) and the cholera toxin B (CTB). While for the infection process 5 identical CTB units assemble to form an annular pentamer and thereby form a pore in the center, the cholera toxin A can infect the cell in question through this pore. An essential prerequisite for the infection process, however, is the contact with the relevant target cell. For this purpose, non-toxic CTB pentamers are used to recruit membrane-bound glycolipid receptors. The preferred targets of cholera toxin B in the process of infection are the ubiquitous on the surfaces of eukaryotic cells occurring GM 1 receptors, wherein at the mGM1 receptor binding with the highest affinity occurs (Galßl, 3GalNAcß1,4 [NeuAca2,3] Galß14GlcCer) (Van Heyningen et al., 1971, Cuatrecasas, 1973). The binding of the cholera toxin to the GM 1 receptors takes place via a so-called binding pocket in the protein structure of the toxin. Upon contact with the receptor, a flexible "loop" in the toxin (amino acids 51-58) causes the cholera toxin to be even more strongly attracted to the receptor through molecular interactions, further stabilizing the toxin-GM1 receptor complex. A further increase in the binding strength is caused by hydrogen bonds to the terminal galactose and sialic acid molecules of the sugar residues of the receptors, resulting in an exceptionally high affinity (dissociation constant: CTB mGM1 receptor: 7.3 × 10 ^-10 mol / L). Cholera toxin also binds (with lower affinity) to other receptors (eg, GD1b, GQ1b, GD1a, GT1b, and GM2) (Kuziemko et al., 1996; Fukuta et al., 1988; Angstrom et al., 1994; Lauer et al., 2002).

After the formation of contact from the cholera toxin to the GM 1 receptor, endocytosis of the complex occurs in the cells. This is done by signal peptides of the cholera toxin using appropriate transporters in the cell membrane. Upon internalization of the cholera toxin, it is taken up into the endoplasmic reticulum via the trans-Golgi complex after retrograde transport (Sandvig et al., 1994, Nichols & Lippincott-Schwartz, 2001). It recruits ARF proteins (ADP-ribosylation factors) to induce irreversible ribosylation of the trimeric GTP-binding protein Gs _a . This results in a permanent activation of adenylate cyclase, which enforces a strong increase of the cAMP level in the cell. The increase in the signal metabolite cAMP then leads to a strong increase in protein kinase A activity, which now causes phosphorylation of many apical chloride channels in the intestinal epithelial cells (Field, 1989). The increased chloride secretion and concomitant blockade of basolateral ion transporters (McRoberts et al., 1985) leads to severe water loss through osmotic mechanisms.

Applications in research and biotechnology (from: Miller, 2008, and Alouf and Popoff, 2006):

1. Distribution of GM-1 Receptors on the Cell Surface and Internalization

The high and very specific binding of CTB to GM 1 receptors is being used in research to tag GM 1 receptors in cellular membranes. As a result, the distribution or occurrence of GM 1 receptors in lipid domains can be determined. Similarly, the dynamics of internalization of GM 1 receptors can be analyzed. By labeling the CTB with fluorescent dyes, the route to internalization, i. which compartments / cell organelles are involved in the cells. In this way, the import into the endoplasmic reticulum of the host cell is documented.

2. Functions of cholera toxin

Due to the non-toxic nature of the CTB subunit, it is used to assess the infection behavior or disease course of cholera enlighten. CTB can be used safely on living cells. By using the CTB subunit, insights into the effects of infection on the expression of genes, the activation of enzymes (eg adenylate cyclase, protein kinase A) and changes in the concentration of signal metabolites (eg cAMP) in the host cell could be analyzed.

3. Determination of cholera toxin content

The measurement of the amount of cholera toxin can be made by quantification via the cholera toxin B subunit. To this end, in an ELISA assay, wells of a plate are coated with catcher molecules (anti-cholera toxin antibodies or GM1 receptors) and brought into contact with the solution to be analyzed. If cholera toxin molecules are contained, they are fixed by the coating antibody or the GM 1 receptor. After addition of another antibody (labeled with enzymes or fluorescent dyes) which is directed against cholera toxin molecules, quantification can take place.

4. Analysis of the distribution of lipid microdomains on the cell surface

The use of biotin or radioactively labeled GM 1 receptors serves to localize so-called lipid microdomains. These sphigolipid and cholesterol enriched regions are visualized with labeled GM 1 receptors so that these microdomains can be screened for cell surface number, size, and distribution. These microdomains are of particular importance for the studies on endo- or transcytosis (for example formation of caveols or further transport as vesicles in the cell).

5. Use of CTB for immunological applications

Cholera toxin was one of the first toxins whose molecular structure and function has been described in detail, as well as its complex immunological properties (protective immunogen, adjuvant and immunomodulator) (Holmgren, 1981, Guerrant, 1985). Among other things, these studies also led to the identification of the non-toxic CTB subunit as a better target for neutralizing antibodies compared to the CTA subunit. Accordingly, the use of CTB as a mucosal vaccine for the induction of intestinal immunity to cholera disease (Holmgren & Svennerholm, 1998). The high stability of CTB as a pentamer thus allows the suitability of this molecule for oral administration. Due to the structural similarity of CTB with the heat-labile enterotoxin from Escherichia coli, immunity is achieved, albeit temporally shorter.

6. Use of the GM 1 receptor for the absorption of enteric viral pathogens

Another use for a GM 1 receptor is the binding or absorption of pathogenic viral particles or enteric viruses in the gastrointestinal tract. Through this application, after oral administration in humans or animals, absorption of viral particles is made possible, so that they can then be eliminated. This is intended to prevent or reduce the infection of enteric viruses. Possible administration forms are the administration of GM1 receptors alone or coupled to nonabsorbable beads (US Pat. No. 5,195,551, May 2, 1989)

The invention aims to achieve a fast and very economical purification of proteins. The object is to develop a method for isolation via fusion proteins. The invention is realized according to the claims.

At the heart of the invention is the utilization of the known high affinity and specificity between GM 1 receptors and cholera toxin B (CTB). GM1 receptor-coated beads or other GM 1 receptor-coated surfaces are used to isolate fusion proteins (which have a CTB domain) from a solution or protein mixture. The binding of the GM1 receptors with the CTB domain of the fusion protein, the separation of other proteins or from mixtures of substances is carried out highly specific.

This also results in the possibility of isolating a fusion protein that contains as component of the amyloid precursor protein (or the amino acid sequence of the binding site or a truncated piece of peptide with the binding site in the amyloid precursor protein for binding to a GM1 receptor ). Efficient binding to the GM1 receptors also takes place here. Of course, other toxins that are used as the domain of a fusion protein may be compatible with the specific / complementary to these toxins Receptors are bound and thus can be cleaned / isolated analogously as in 1. A peculiarity of toxins that bind to suitable receptors is the high affinity and specificity of the binding that are suitable for the purpose shown here.

The decisive advantage of our development lies in the avoidance of the use of antibodies on the one hand and in the large time savings on the other, as shown below.

Many research groups around the world use antibodies to enrich or isolate proteins. The preparation of the necessary antibodies (for each protein must be carried out a separate generation of the corresponding antibodies) almost always animals must be used. These are vaccinated several times, so that after several weeks sufficient quantities of specific antibodies can be extracted from the serum. Animal husbandry costs are incurred (especially large mammals such as sheep, donkeys or goats are very cost intensive) and often end in death, which is particularly the case in smaller mammals to recover all the blood and antibodies contained therein. Another problem is also the difference in quality with respect to the specificity of the antibodies directed against the antigen, which is often very variable from animal to animal. Furthermore, with this method, even in the same animal no uniform antibody population is generated, but only a mixture of different antibodies against different epitopes of the antigen (so-called polyclonal antibodies), so that re-immunizations of other, additional animals are often required. In addition to the polyclonal antibodies, there is also the method of producing monoclonal antibodies, which can then ensure a consistent quality for a long time. But here, too, animals must be used, which after repeated vaccinations, the spleen is removed to perform a merger with hybridoma cells. However, these are extremely labor-intensive and cost-intensive procedures. The establishment of a monoclonal antibody-producing cell line for a single antigen or target protein usually costs not less than € 10000 feasible.

Other possibilities of protein isolation include the use of methods such as gel filtration, ion exchange chromatography, salting out or chromatography methods based on hydrophobic interactions. An attempt is made, by using individual chemical / biochemical or physical Characteristics of the target proteins to separate them from unwanted proteins. Frequently, different procedures have to be coupled with it. The testing or adaptation of the method is not only very time-consuming and thus costly, but also inevitably leads to an increasing loss of the target product during processing.

In the method we have described, the effort and risk of success in isolation is very limited. Preparatory takes place only the introduction of a gene (it contains the DNA sequence for the CTB domain and the target protein and, where appropriate, the objectives of the work, a protease interface between the DNA sequence of the CTB domain and the target protein) in an expression organism (eg Yeasts, bacteria or plants). The necessary processes are often practiced in molecular biology and well-known or well-controlled methods. During the protein synthesis, which naturally takes place permanently in the organism, the fusion protein which is cloned into the organism via a gene is also produced, which is separated from the other unwanted proteins / substances by the GM1 receptors (commercially commercially available).

The invention will be explained in more detail by application examples.

Application examples:

1. Isolation of a fusion protein for immunization by permanent

Fixation to a mobile matrix:

The purification of proteins, among other things, for immunization in a high purity is very often associated with many substeps, which in sum are not only time and cost intensive, but also lead to losses in the yield of the product compared to the existing starting amount. We minimize these losses and accelerate the recovery of target proteins by binding GM 1 receptors to biodegradable beads and applying them to a protein mixture of genetically modified organisms. The gene sequence for the fusion protein (consisting of the cholera toxin and the desired target protein) was cloned into these organisms by means of gene vectors, so that the genetically modified organisms express this fusion protein. The addition of the beads coated with the GM1 receptors leads to the formation of complexes consisting of (biodegradable) beads + GM1 receptors + fusion proteins, which can be reliably, easily and quickly separated from other unwanted proteins with just a few washes. Due to the fact that the CTB domain has an adjuvant, immunogenicity-enhancing effect, this complex can be used to immunize against diseases.

2. Isolation of a Fusion Protein Without Permanent Fixation to a Matrix The purification or isolation of the fusion protein takes place in the same way as in Application Example 1. An extension of this method is that after binding of the GM1 receptor to the fusion protein, the entire complex is detached from the matrix again becomes. Thus, the entire complex can be resumed in solution. For this purpose, reagents are used which allow the binding of the receptor from the matrix.

Another additional possibility of removing the entire complex from the matrix can be carried out in the following manner: Holmberg et al. described the possibility of a separation of bound biotin and streptavidin molecules after short-term exposure to heat in aqueous systems (Holmberg et al., 2005). This effect, described by Holmberg, is used for the separation of the GM1 fusion protein complexes from the matrix. Using biotinylated GM 1 receptors and magnetic beads (coated with streptavidin), fusion proteins can be bound to a cholera toxin domain. After separation of the magnetic (streptavidin-coated) beads with the (biotinylated) GM1 fusion protein complexes bound thereto from the remaining proteins, the separation of the matrix (s) by heat can now be effected by breaking the bond between biotin and streptavidin becomes. In order to keep the time of the heat impact on the fusion protein as low as possible (protection against possible denaturation by thermal effects), the beads with the coupled GM1 receptor fusion protein complexes flow through a thin capillary / tube in an aqueous medium. Due to this small diameter, the solution can be heated very rapidly at one point and then cooled again (because the heated volume of the solution is very small) in order to minimize the risk of denaturation of the fusion protein. The energy supply can, for example, by an infrared laser. Likewise, an energy source for heat generation in the form of an electromagnetic alternating field (induction field) can be used. In this case, only the magnetic beads are heated by the induction field, so that the risk of denaturation of the fusion protein can be further minimized, because less the aqueous medium but primarily the matrix (magnetic beads) is heated. After separation of the magnetic (streptavidin-coated) beads from the biotinylated GM1 receptors (with the fusion protein complexes bound to them), re-restoration of streptavidin-biotin binding must be prevented. This can be done by a strong extension of the Kapillar- / Rohrquerschnitts together with a sheath of the widened / enlarged capillary (or tube) with a ring magnet (or other device for generating a magnetic field) immediately after the point at which in the Capillary the energy supply for heat generation takes place (see Figure 2/2). The result is the following effect: While the separated (biotinylated) GM 1 receptors with the bound fusion protein complexes are transported further with the liquid flow, the (streptavidin-coated) beads remain on the insides of the capillary walls enclosed by the magnet.

The free GM1 fusion protein complexes can be used in emulsion for immunization purposes, but it is also possible to obtain native enzymes for scientific, industrial or medical purposes (eg restriction nucleases, proteases, enzymes for syntheses), but it can also be a preservative for longer-term storage (Freeze-drying). With application example 2 (using a GM1 receptor-coated, planar matrix), after incubation of a protein extract from plants (containing the expressed fusion protein) and only three final washings, a purity of at least 95% can be achieved (control was carried out by separation of the detached Complexes of the matrix in SDS-PAGE followed by silver staining)

3. Isolation of a fusion protein with a protease cleavage site

A further development of this quick and easy purification consists in the subsequent separation of the cholera toxin domain from the remaining part of the Fusion protein (target protein domain). For this purpose, before cloning the fusion protein gene into the expression vector, a gene sequence for a protease cleavage site is inserted between the sequence coding for the cholera toxin and the "attached" (target) protein. After synthesis of the fusion protein with the protease cleavage site in a corresponding expression system, as in application example 1, the separation from the other proteins or mixtures of substances takes place. By adding a protease suitable for the interface, the complex fixed on the matrix (consisting of:

GM1 receptor + cholera toxin domain + protease interface + target protein domain) the part of the functional protein can be separated. If the protease used is previously biotinylated, the protease can be removed again using streptavidin-coated beads (corresponding kits consisting of biotinylated protease and streptavidin-coated beads are commercially available). The functional protein in solution after separation can subsequently be concentrated or concentrated with molecular sieves. The remaining on the matrix GM 1 receptors with the bound cholera toxin domain are completely discarded or the matrix is regenerated.

source

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Angström J, Teneberg S, Karlsson KA .: Delineation and comparison of ganglioside-binding epitopes for the toxins of Vibrio cholerae, Escherichia coli, and Clostridium tetani: evidence for overlapping epitopes. Proc Natl Acad. See U S A. 1994 Dec 6; 91 (25): 11859-63.

Clemens JD, Sack DA, Harris JR, Chakraborty J, Neogy PK, Stanton B, Huda N, Khan MU, Kay BA, Khan MR, et al .: Cross-protection by B subunit-whole cell cholera Vaccine against diarrhea associated with heat -labile toxin-producing enterotoxigenic Escherichia coli: results of a large-scale field trial. J Infect Dis. 1988 Aug; 158 (2): 372-7.

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Fukuta S, Magnani JL, Twiddy EM, Holmes RK, Ginsburg V .: Comparison of the carbohydrate-binding speeificities of cholera toxin and Escherichia coli heat-labile enterotoxins LTh-1, LT-Ila, and LT-IIb. Infect immune. 1988 Jul; 56 (7): 1748-53.

Holmberg A, Blomstergren A, Nord O, Lukacs M, Lundeberg J, Uhlen M .: The biotin-streptavidin interaction can be reversibly broken using water at elevated temperatures. Electrophoresis. 2005 Feb; 26 (3): 501-10.

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List of Figures

Figure 1: Schematic representation of the purification of a protein by isolation via a toxin binding domain and the cleavage by a protease

Figure 2: Schematic representation of the isolation of a fusion protein without permanent fixation to a matrix

Claims

claims

1. A process for the rapid isolation or purification of recombinant proteins with a (non-toxic) cholera toxin domain, characterized in that a matrix is coated with GM1 receptors and brought into contact with a protein extract, which is a recombinant protein consisting of a Choleratoxinproteindomäne and a protein to be purified, after binding of the recombinant protein to the matrix is washed several times with buffer and finally carried out a further processing of the resulting protein.

Method according to claim 1, characterized in that a matrix e.g. consists of beads (beads) of size from 1 μηι to 100 μηι.

3. The method according to claim 1, characterized in that the fusion protein is detached from the matrix.

4. The method according to claim 1, characterized in that the fusion protein is further processed together with the matrix.

5. The method according to claim 1, characterized in that the fusion protein either on the matrix or after detachment from the matrix by suitable

Proteases is split.

6. The method according to claim 1 to 4, characterized in that the

Fusion protein, optionally after isolation, for the immunization of

Diseases is used.