WO2013026445A1 - Procédé pour l'isolement ou la purification rapide de protéines de recombinaison comprenant un domaine de la cholératoxine (non toxique) - Google Patents

Procédé pour l'isolement ou la purification rapide de protéines de recombinaison comprenant un domaine de la cholératoxine (non toxique) Download PDF

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Publication number
WO2013026445A1
WO2013026445A1 PCT/DE2012/000877 DE2012000877W WO2013026445A1 WO 2013026445 A1 WO2013026445 A1 WO 2013026445A1 DE 2012000877 W DE2012000877 W DE 2012000877W WO 2013026445 A1 WO2013026445 A1 WO 2013026445A1
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Prior art keywords
protein
cholera toxin
fusion protein
matrix
binding
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PCT/DE2012/000877
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German (de)
English (en)
Inventor
Holger PRIETZSCH
Juliane KLÜSS
Alain Steinmann
Udo Meyer
Nadine KOLP
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Bioserv Analytik Und Medizinprodukte Gmbh
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Publication of WO2013026445A1 publication Critical patent/WO2013026445A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the complex immunological properties were investigated (protective immunogen, effective mucosal adjuvant, immunomodulator).
  • CTB non-toxic subunit B
  • CTB non-toxic subunit B
  • 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 is the contact with the relevant target cell.
  • 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.
  • a flexible "loop" in the toxin 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).
  • 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 .
  • ARF proteins ADP-ribosylation factors
  • CTB CTB to GM 1 receptors
  • GM 1 receptors GM 1 receptors in cellular membranes.
  • the distribution or occurrence of GM 1 receptors in lipid domains can be determined.
  • the dynamics of internalization of GM 1 receptors can be analyzed.
  • 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.
  • CTB subunit 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.
  • enzymes eg adenylate cyclase, protein kinase A
  • cAMP signal metabolites
  • the measurement of the amount of cholera toxin can be made by quantification via the cholera toxin B subunit.
  • cholera toxin B subunit a plate 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.
  • GM 1 receptors serve 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).
  • 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.
  • GM 1 receptor 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.
  • 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.
  • CTB cholera toxin B
  • 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.
  • 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 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).
  • an expression organism eg Yeasts, bacteria or plants.
  • the necessary processes are often practiced in molecular biology and well-known or well-controlled methods.
  • 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 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.
  • 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.
  • fusion proteins can be bound to a cholera toxin domain.
  • the separation of the matrix (s) by heat can now be effected by breaking the bond between biotin and streptavidin becomes.
  • 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.
  • an energy source for heat generation in the form of an electromagnetic alternating field (induction field) can be used.
  • induction field electromagnetic alternating field
  • 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.
  • 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).
  • native enzymes for scientific, industrial or medical purposes eg restriction nucleases, proteases, enzymes for syntheses
  • Freeze-drying Freeze-drying
  • 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).
  • 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.
  • Shiga B-fragment is regulated by butyric acid and cAMP. J Cell Biol. 1994 Jul; 126 (1): 53-64.
  • 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

Abstract

L'invention concerne un procédé pour la purification rapide, efficace et simple de protéines issues de mélanges de protéines ou de matières. On a recours à la liaison d'une toxine (comme la cholératoxine) au récepteur correspondant (comme le récepteur GM1), qui s'effectue non seulement de manière très spécifique, mais également avec une affinité élevée. Ces propriétés de la liaison (affinité, spécificité) sont utilisées dans cette invention, du fait qu'une protéine de fusion est exprimée, laquelle possède un domaine de liaison à la toxine. La protéine de fusion peut être utilisée en totalité ou seulement en partie (sans domaine de liaison à la toxine) après clivage par une protéase en un emplacement défini. La présente invention vise à proposer une purification rapide et très économique pour des protéines. Les domaines d'application sont la médecine, la médecine vétérinaire, la science et la recherche ainsi que différentes branches de l'industrie.
PCT/DE2012/000877 2011-08-24 2012-08-24 Procédé pour l'isolement ou la purification rapide de protéines de recombinaison comprenant un domaine de la cholératoxine (non toxique) WO2013026445A1 (fr)

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DE201110111688 DE102011111688A1 (de) 2011-08-24 2011-08-24 Verfahren zur schnellen Isolation bzw. Aufreinigung von rekombinanten Proteinen mit einer (nicht toxischen) Choleratoxin-Domäne
DE102011111688.9 2011-08-24

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Citations (4)

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Publication number Priority date Publication date Assignee Title
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US5192551A (en) 1989-05-02 1993-03-09 Johns Hopkins University Neutral glycolipid as an adsorbent for enteric viral pathogens
US5268276A (en) * 1988-09-16 1993-12-07 Jan Holmgren Recombinant systems for expression of cholera B-sub-unit with the aid of foreign promoters and/or leader peptides

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0368819A1 (fr) * 1988-09-16 1990-05-16 Aktiebolag Vitec Expression de la sous-unité liante de la toxine chloérique à l'aide de promoteurs étrangers et/ou de séquences guides
US5268276A (en) * 1988-09-16 1993-12-07 Jan Holmgren Recombinant systems for expression of cholera B-sub-unit with the aid of foreign promoters and/or leader peptides
US5192551A (en) 1989-05-02 1993-03-09 Johns Hopkins University Neutral glycolipid as an adsorbent for enteric viral pathogens
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