DE10307793A1 - Method for detecting and removing endotoxins, useful for treating e.g. recombinantly produced pharmaceuticals or nucleic acid, by incubation with phage tail protein - Google Patents

Abstract

Method for detecting endotoxins (I) comprises incubating a sample with a bacteriophage tail protein (II), then detecting any (I) bound to (II). An independent claim is also included for a method of removing (I) from a sample by incubation or contact with (II) that is immobilized, non-specifically or in a targeted manner, on a solid carrier.

Description

The present invention relates to a method for the detection and depletion of endotoxins a sample.

Endotoxin (ET) means a family of lipopolysaccharides, along with proteins and phospholipids the outer cell wall Form gram-negative bacteria. Endotoxins only come in this Bacterial group and play an important role in the organization, stability and barrier function of the outer membrane. Numerous bacteriophages use endotoxin or lipopolysaccharide in general for the specific detection of their host bacteria.

All endotoxin variants exist from a heteropolysaccharide that is covalently bound to lipid A. (Holst, O., 1999, Chemical structure of the core region of lipopolysaccharides. In: Endotoxin in health and disease (Brade, H., Morrison, D.C., Opal, S., Vogel, S. eds.), Marcel Dekker Inc. New York)). lipid A anchors endotoxin in the outer bacterial membrane. The heteropolysaccharide, which consists of a heart oligosaccharide and the O-antigen exists, points into the surrounding solution and determines the serological identity of the bacterium. The O-antigen consists of repetitive oligosaccharide units, whose composition is strain-specific (see Holst et al., supra). Characteristic building blocks of cardiac oligosaccharide are 2-keto-3-deoxyoctonic acid (KDO) and L-glycero-D-manno-heptose (Hep).

The most conservative part of endotoxin Lipid A is of various genera. Preserved similarly to lipid A is the inner heart region, the outer heart region already shows a higher one Variation on. Wear the inner heart region, KDO and Lipid A yourself several phosphate groups as substituents and are so for the negative Charge of endotoxin responsible. In addition, the Phosphate groups on lipid A and the heart region variable with arabinose, Ethanolamine and phosphate can be substituted. Individual saccharide building blocks of the O-antigen are acetylated, sialysed or glycosylated. The O-antigen also varies regarding the Number of repetitive units, which is why the endotoxin population each Bacterium has a certain heterogeneity (Palva E. T., Makela P.H., Lipopolysaccharides heterogeneity in Salmonella typhimurium analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Eur J Biochem. 1980; 107 (1): 137-43; Goldman R.C., Leive L., Heterogeneity of antigenic-side-chain length in lipopolysaccharide from Escherichia coli 0111 and Salmonella typhimurium LT2., Eur J Biochem. 1980; 107 (1): 145-53).

Endotoxins are biomolecules that without appropriate precautions in practically all watery ones solutions are to be found. Endotoxins can in humans and animals to sepsis, a strong malfunction of the immune system to lead. Therefore e.g. impurities in the manufacture of pharmaceutical proteins Detect endotoxin exactly and subsequently remove it completely. Endotoxin poses a problem with genetically engineered drugs Gene therapy drugs or substances that are found in humans or animals (e.g. veterinary Treatment or in animal experiments). But not only in medical, but also in research applications, such as inhibition may occur during mammalian transfection experiments or a decrease in transfection efficiency by endotoxin was observed become.

To proteins in the context of clinical To be able to use studies demand the European and the American Pharmacopeia that the proteins determined Below the limit values for endotoxin exposure (e.g. immune serum Globulin 0.91 EU / ml, this corresponds to 5 EU / kg body weight & hour (dose = EU / kg · h); EU = endotoxin unit; FDA (Food and Drug Administration): Guideline on Validation of LAL as End Product). If a drug or proteins contained therein may have too high an endotoxin load this will lead to the death of the subject. The misguided immune system damages through an overreaction the patient. This can lead to tissue inflammation, a drop in blood pressure, rapid heartbeat, Lead to thrombosis, shock etc. Already a longer one Prolonged exposure to endotoxins in picogram amounts can become chronic Side effects such as Immunodeficiency, septic symptoms etc. lead. In the context of substance production, therefore, especially in processes under “Good Manufacturing Practice "(GMP) Conditions, trying to deplete endotoxin as much as possible. Indeed is the endotoxin removal from proteins, polysaccharides and DNA problematic. There are big problems with proteins in particular through their intrinsic properties such as state of charge or hydrophobicity, the one Almost prevent endotoxin removal or lead to large product losses lead in the removal procedure can.

There are currently only three procedures for Endotoxin detection in biological solutions is described, whereby only the first two procedures are approved by the FDA. 1. "Rabbit Pyrogen Testing ": A process at which injected an endotoxin solution into a live rabbit and thus an Immune response is triggered. This endotoxin-induced immune response is about the development of fever demonstrated. 2. The “Limulus Amoebocyte Lysate (LAL) "test, the most used currently Test (BioWhittacker, Inc., Charles River, Inc., Associates of Cape Cod, Inc., all U.S.). With this procedure, the clumping the blood of horseshoe crab (Limulus polyphemus) after contact with endotoxin measured. 3. Another way is the use of a special cell culture system (Sterogene Inc., USA) with which the activation of monocytes on the emergence of certain Cytokine is tracked.

However, the first two methods are very expensive (cf. Endoto competitor comparison xin detection) and due to the great need for experimental animals and blood of the very rare horseshoe crab, not least because of animal welfare reasons. Although the LAL test can also be miniaturized and automated, it has massive disadvantages in use due to the low stability of the components. An open LAL solution has to be processed and used immediately, since the components aggregate within a few hours. Trained personnel are required for all test procedures and the procedures are very susceptible to interference because, for example, rabbits' immune systems can react differently to the same endotoxin dose. The cell culture process from Sterogene, like all cell culture processes, is also very complex and has problems with standardization.

Overall, it can be determined that it is not an easy-to-use, inexpensive method for endotoxin detection there and the methods currently used have a number of disadvantages exhibit. There is therefore a need for a method that does this Avoids disadvantages.

For endotoxin depletion from biological solutions there are generally a number of procedures. Especially with proteins so far, however, there are no generally applicable standard procedures. The methods used are adapted to the specific Properties of the respective protein and on the corresponding Production process of the protein. There are different possibilities for endotoxin depletion, each of these methods being specific Has advantages and disadvantages.

Ultrafiltration (Petsch, D. & Anspach, F. B., 2000, J. Biotechnol. 76, 97-119 and references in it) is for Endotoxin depletion from water and solutions with low molecular weight Ingredients such as salts, sugar and antibiotics are used, however not for high molecular weight proteins or DNA.

The 2-phase extraction (e.g. WO 0166718, Merck) is said to be water soluble Separating proteins and DNA from endotoxin, however, requires detergent residues in the cleaned product. The procedure is also repeated several times the cleaning procedure is time consuming.

An anion exchanger (DEAE) process is also used for the endotoxin depletion from DNA and basic proteins (e.g. US 5990301 , Qiagen; WO 9414837, Enzon), which, however, requires a low ionic strength (<50 mM NaCl) and leads to protein co-adsorption with acidic proteins.

Another method for endotoxin depletion from DNA and proteins (e.g. BSA, myoglobin, gamma-globulin, cytochrome C) is affinity adsorption (e.g. polymyxin B, histamine, histidine, polylysine), for example GB 2192633 (Hammersmith Hospital), which, however, is toxic in the case of polymyxin B and can lead to co-adsorption of proteins at low ionic strengths.

Immum affinity chromatography is also used, whereby the specificity for certain endotoxins can only be achieved using expensive antibodies ( US 5179018 , Centocor; WO 0008463, Bioserv) against cardiac oligosaccharide can be achieved.

The S3 delta peptide (WO 0127289) of factor C (a component of the LAL test) (WO 9915676 both: National University of Singapore) for proteins (e.g. BSA, Chymotrypsinogen), but this method is a has low efficiency at high ionic strengths and the high Manufacturing costs (production in insect cell culture) are added.

• – Anionenaustauscherchromatographie
• – Reversed-Phase Chromatographie; Diese hat den Nachteil, dass sie nicht für alle Proteine gleichermaßen geeignet, – insbesondere bei hydrophoben Proteinen problematisch ist. Darüberhinaus ist dieses Verfahren sehr zeitintensiv.
• – RemTox (Fa. Millipore): Dieses Verfahren hat den Nachteil, das neben einer sehr langen Inkubationsdauer, der unspezifische Bindungsanteil hoch ist, und die Proteinwiederfindung oftmals nicht ausreichend ist.

There are essentially three methods used in the pharmaceutical industry for protein solutions, adapted to the properties of the target proteins:

• - Anion exchange chromatography
• - Reversed phase chromatography; The disadvantage of this is that it is not equally suitable for all proteins, and is particularly problematic with hydrophobic proteins. In addition, this process is very time-consuming.
• - RemTox (Millipore): This method has the disadvantage that, in addition to a very long incubation period, the non-specific proportion of binding is high, and protein recovery is often not sufficient.

A rough endotoxin depletion from proteins to a value up to 10 EU / ml is with the existing ones Procedure in many cases possible. However, the remaining concentration of endotoxin is still effective toxic. A further depletion (= fine cleaning) is therefore necessary or dependent from the dose of the protein in medical application, from the European Pharmacopeia (e.g. 5 EU / kg body weight and Hour in intravenous Applications) and the FDA. Indeed this fine cleaning is often unsatisfactory with existing methods guaranteed. The marketable Processes have considerable disadvantages here and are specific Proteins often do not, or only with considerable loss of the target protein, applicable.

Therefore, the object of the invention based on providing a method that samples endotoxins can demonstrate. The invention is also based on the object to provide a method by which endotoxins from aqueous solutions can be removed.

The tasks are carried out by the in the patent claims defined object solved.

The following figures explain the Invention.

1 shows a schematic overview of the chemical structure of endotoxin from E. coli O111: B4. Hep = L-glycero-D-mannoheptose; Gal = galactose; Glc = glucose; KDO = 2-keto-3-deoxyoctonic acid; NGa = N-acetyl-galactosamine; NGc = N-acetylglucosamine.

2 shows the results of experiments with chromatography columns carrying NStrepS3Cp12 immobilized via sulfhydryl residues. (A) Endotoxin removal from protein solutions: bovine serum albumin (BSA), carbon anhydrase (CA) and lysozyme (Lys) were incubated on the column for 1 h and then eluted with buffer. The endotoxin concentration before and after the column was measured with the LAL test and the percentage decrease was calculated therefrom. (B) Protein recovery: The protein concentrations of the starting solutions and the fractions after the column were determined by absorption measurement at 280 nm and the percentage protein recovery was determined therefrom.

3 shows the endotoxin removal from a lysozyme solution via chromatography columns with "undirected" (1) and "directed" (2) immobilized p12. In both cases, p12 S3C was bound to NHS-activated columns. The “non-directional” immobilization was carried out via primary amino residues of p12S3C, which form covalent compounds with the carrier substance by reaction with the NHS groups. A “directional” linkage of p12S3C via an N-terminal cysteine is achieved by diaminoethane and SIA (N-succinimidyl- iodoacetate) reached. (A) percentage endotoxin removal. (B) protein recovery.

4 shows the results of experiments with biotinylated p12 which was bound to magnetic beads via streptavidin. (A) The endotoxin depletion from buffer (20 mM Hepes, 150 mM NaCl, pH 7.5) and protein solutions was determined using the LAL test. (B) For the protein solutions, protein recovery was determined by absorption measurements. The beads were separated from the solution using a magnetic separator. BSA: bovine serum albumin. CA: carbon anhydrase. Lys: lysozyme.

5 shows the results of endotoxin removal with p12, which was immobilized on agarose beads via biotin-streptavidin interactions. The immobilized p12 was removed by centrifugation. The endotoxin removal from buffer (20 mM Tris, 150 mM NaCl, pH 8.0) and BSA solutions was determined on the basis of the endotoxin concentrations of the starting solution and the supernatant.

gemessen wurden. Die Bindung erfolgt an Endotoxin von E. coli D21fl, das auf einem hydrophoben HPA-Chip immobilisiert wurde. Die Injektion von p12 und EDTA (5 mM) wird durch Balken über den Kurven markiert. Puffer: 20 mM Tris, 150 mM NaCl, pH 8.0. (B) Gleichgewichtsresonanzwerte für die Bindung von p12 an immobilisiertes Endotoxin wurden etwa 600 s nach Beginn der p12 Injektion gemessen und gegen die dazugehörigen p12-Konzentration aufgetragen. Die durchgezogene Linie zeigt einen Fit der Langmuirsche Adsorptionsisotherme (RU = RU_max·[p12]/([p12] + K_d)) an die Daten. (C) Bindung von E. coli an biotinyliertes p12, das auf Streptavidin-Chips immobilisiert wurde. E. coli D21e8

dessen innerere Herz-Region vollständig ist, an p12. Dagegen bindet E. coli D21f2 (----), der eine stark verkürzte Herz-Region besitzt, bindet nicht an p12. Die Messungen wurden in PBS durchgeführt. 6 shows results of surface plasmon resonance measurements. (A) Resonance curves in response to injection of different (each in μg / ml: 100; 25; 6.25; 4; 1.56; 0.4) p12 concentrations

were measured. Binding occurs to endotoxin from E. coli D21fl, which was immobilized on a hydrophobic HPA chip. The injection of p12 and EDTA (5 mM) is marked by bars above the curves. Buffer: 20 mM Tris, 150 mM NaCl, pH 8.0. (B) Equilibrium resonance values for the binding of p12 to immobilized endotoxin were measured about 600 s after the start of the p12 injection and plotted against the associated p12 concentration. The solid line shows a fit of the Langmuir adsorption isotherm (RU = RU _max · [p12] / ([p12] + K _d )) on the data. (C) Binding of E. coli to biotinylated p12 that was immobilized on streptavidin chips. E. coli D21e8

whose inner heart region is complete, at p12. In contrast, E. coli D21f2 (----), which has a strongly shortened heart region, does not bind to p12. The measurements were carried out in PBS.

7 shows schematically the structure of the endotoxin heart region of various E. coli mutants.

8th shows schematically the result of an endotoxin depletion using a chromatography column flow-through method. E means equilibration buffer (20 mM Hepes, 150 mM NaCl, 0.1 mM CaCl ₂ , pH 7.5), A means wash buffer A (20 mM Hepes, 150 mM NaCl, 0.1 nM CaCl ₂ , pH 7.5), B means elution buffer B (20 mM Hepes, 150 mM NaCl, 2 mM EDTA, pH 7.5), C means regeneration buffer C (20 mM Hepes, 150 mM NaCl, 2 mM EDTA, 0.005% NaDOC, pH 7.5), S means concentration of protein and endotoxin in the starting solution. BSA means bovine serum albumin. EU means Endotoxin Units. After injection (I) of 4 ml of the starting solution (S), rinsing was carried out with 15 ml of washing buffer and the run was fractionated (2.5 ml each during application, 2 ml each during washing). The column was then regenerated with buffers B and C and the outlet was also collected in fractions (2 ml each). As can be seen in the figure, the BSA was found in the first 3-5 fractions after the injection. The endotoxin content in these fractions was 100 times lower than in the starting solution. The endotoxin bound to the column was then washed with buffers B and C from the column.

9 shows schematically the results of the endotoxin removal from slightly contaminated buffer solution (5 EU / ml) in the flow method. p12 was immobilized on NHS-activated Sepharose 4 FastFlow (Amersham Biosciences, Uppsala, Sweden) (8 mg p12 / 1 ml Sepharose) and poured into 3 columns with 2 ml column volume each. The experiment was carried out in parallel on 3 columns. Before the sample was applied, 1 ml of equilibration buffer (20 mM Hepes, 150 mM NaCl, 0.1 mM CaCl ₂ , pH 7.5) was collected, then the sample (S: endotoxin from E. coli O55: B5 in equilibration buffer, 4.6 EU / ml ) injected (I) and fractions of 5 ml and 2 ml collected. The column was regenerated by adding 4 ml of regeneration buffer (B: 20 mM Hepes, 150 mM NaCl, 2 mM EDTA, 0.005% NaDOC, pH 7.5). The endotoxin concentration was determined using the LAL test (kinetically chromogenic LAL test, Charles River Inc.). The endotoxin impurities could be completely removed in all three experiments, ie the endotoxin concentration in the run was below the detection limit (<0.005 EU / ml).

The term "endotoxin depletion" as used herein means complete or partial removal of endotoxin from sample material.

The term "endotoxin" as used herein means bacterial Lipopolysaccharide, the component of the outer membrane gram-negative Bacteria.

The term "bacteriophage tail protein" as used herein denotes those proteins that occur in bacteriophages and Can bind components of cell membranes. Usually these are proteins localized in the bacteriophage tail, but can also on the bacteriophage head or localized in bacteriophages without tail on the normal bacterial envelope his. The cellular components bound by the bacteriophage tail protein recognize in particular endotoxins.

The term "non-specific immobilization" or "non-directional immobilization" as used here means that the coupling of a protein to a matrix via protein residues (e.g. primary amines) takes place over the entire protein surface are distributed. The selection of for the coupling of the single protein molecule used group is random.

The term "directional immobilization" as used here means coupling over amino acid residues or other residues (e.g. glycosylation of the protein), whose position in the protein (e.g. N- or C-terminal) is known. Choosing these groups for the coupling takes place through the selection of suitable reaction partners / linkers, who prefer to react with these residues (e.g. coupling of sulfhydryl residues on iodoacetate residues; Indoacetate reacts a thousand times faster Sulfhydryl residues than with amino residues).

• a) Inkubieren einer Probe mit einem Bakteriophagenschwanzprotein,
• b) Nachweis von an Bakteriophagenschwanzproteine gebundenes Endotoxin.

The present invention relates to a method for the detection of endotoxin, comprising the steps:

• a) incubating a sample with a bacteriophage tail protein,
• b) Detection of endotoxin bound to bacteriophage tail proteins.

The invention preferably relates a method in which the detection by means of spectroscopic methods, e.g. Fluorescence emission, fluorescence polarization, absorption or Circular dichroism, or by means of capacity measurement, e.g. electrical Signals, or carried out indirectly by means of proof of competition.

If necessary, after step a) and before step b) an additional one Step a ') separation of bacteriophage tail protein-endotoxin complex from the sample introduced.

• a) Inkubation oder in Kontakt bringen einer Probe mit Bakteriophagenschwanzproteinen, die unspezifisch oder gerichtet, an einem festen Träger immobilisiert sind,
• b) Trennen des Bakteriophagenschwanzprotein-Endotoxin-Komplexes von der Probe.

The present invention further relates to a method for removing endotoxin from a sample, comprising the steps:

• a) incubation or contacting a sample with bacteriophage tail proteins, which are non-specific or directed, immobilized on a solid support,
• b) separating the bacteriophage tail-protein-endotoxin complex from the sample.

Before the incubation, the ion composition of the divalent ions, for example Ca ²⁺ , Mg ²⁺ and / or the pH is preferably adjusted in order to obtain an optimal endotoxin-bacteriophage tail protein binding. Also preferred during or after the incubation is a “unmasking” of the bound endotoxin by adding detergents and / or salts, for example Tween, Triton, NaCl or ammonium sulfate, or other substances, for example chitosan, sugar or lipids, which detach the endotoxins from eg accelerate proteins or nucleic acids.

The bacteriophage tail protein can be a naturally occurring one or a molecular biological or biochemically modified one. The bacteriophage tail protein can be genetically and / or biochemically modified for various reasons. However, not only the naturally occurring bacteriophage tail proteins can be used for the methods according to the invention, but also their variants. For the purposes of the present invention, variants means that the bacteriophage tail proteins have a modified amino acid sequence. These can be obtained by screening the naturally occurring variants, or by random mutagenesis or targeted mutagenesis, but also by chemical modification. The bacteriophage tail proteins used for the methods according to the invention can be adapted to carrier structures in their specificity or their binding properties by means of targeted or random mutagenesis. This bond to the carrier can be fixed, for example covalently or via a specific or non-specific biotinylation, but can also be reversible, for example via a reducible disulfide bridge. Furthermore, the stability can be increased by a modification. Molecular biological or chemical mutagenesis introduces mutations, which can be amino acid additions, deletions, substitutions or chemical modifications. These mutations can cause a change in the amino acid sequence in the binding region of the bacteriophage tail proteins with the aim of adapting specificity and binding affinity to test needs, for example increasing the binding of the endotoxins to the bacteriophage tail proteins or making them irreversible in order to improve detection or depletion. In addition, a genetic engineering or biochemical modification of the phage proteins can be carried out with the aim of switching off any enzymatic activity which may be present, in order to improve the binding or to make it irreversible. Furthermore, genetic engineering or chemical modification of the phage proteins can be carried out in order to determine the physical properties of the protein, such as solubility, thermal stability, etc., in the sense of the invention Adapt the procedure.

Work to educate the three-dimensional structure of T4 p12 had shown that at elevated temperature 33 kDa and 45 kDa proteolytic fragments can be generated N- and C-terminal (33 kDa) or only N-terminal (45 kDa) are shortened. In contrast to the 33 kDa fragment, the 45 kDa fragment is still able to bind to bacteria. Hence, the C-terminus involved in cell binding.

The modification can also in particular have the purpose of providing direct evidence e.g. by measuring the Allow tryptophan fluorescence. For example, P12 has five Tryptophan residues. The fluorescence spectrum of the native protein indicates indicates that these residues are largely solvent-inaccessible. It is known from a large number of scientific papers that almost always aromatic amino acids bind sugar residues, as they also occur in endotoxin. The connection the sugar residues on proteins can be quenched by trypthophane fluorescence, or, if necessary, additionally through a change of the fluorescence maximum are tracked. Let your own work suspect the inconvenient Distribution of the fluorophores of the natural p12 an exploitation which prevents fluorescence properties of p12 for binding measurement. The fluorescence properties of p12 are due to the five tryptophan residues dominates, whose fluorescence cannot be measured by the addition of endotoxin changed becomes. These data suggest that tyrosine residues are more likely than tryptophan residues are involved in the binding, their signal change against the high tryptophan background cannot be made visible. Based on the proteolysis results come six tyrosines at the C-terminus of p12 for the endotoxin detection kit into question, which can be made "visible" accordingly a selective molecular biological exchange of the five tryptophan residues the first step against tyrosine is the spectroscopic Properties so deliberately changed that endotoxin binding by fluorescence signal change of a single tryptophan residue is measurable. Subsequently through a targeted exchange of one of the six Tyrosine in the C-terminal area against a tryptophan residue intensity of the measurable signal increased significantly for the development of an endotoxin detection kit to get attractive signal differences.

What bacteriophage tail proteins used depends depends on which endotoxins are detected or purified should. A large number of known bacteriophages already stand for one large part of the bacteria described so far are available and can be used for the method according to the invention be used. The phages and the corresponding host bacteria include available from the following master collections: ATCC (USA), DSMZ (Germany), UKNCC (Great Britain), NCCB (Netherlands) and MAFF (Japan).

The bacteriophage tail proteins are preferably derived for the inventive method of Bacteriophages, their host bacteria, medically or biotechnologically have relevant meaning, e.g. E. coli that is in production recombinant proteins or nucleic acids used for gene therapy becomes. Particularly preferred are bacteriophage tail proteins that bind highly conserved areas of endotoxin, e.g. the heart region or lipid A. Particularly preferred are p12 and p12-like bacteriophage tail proteins With a combination of endotoxin contaminants from different host bacteria can be a combination of the corresponding endotoxin-detecting bacteriophage tail proteins be used.

Evidence or depletion of endotoxin in or from a sample occurs via the binding of endotoxin to the bacteriophage tail proteins. This bond can e.g. by direct measurement using spectroscopic methods, e.g. about fluorescence emission, Fluorescence polarization, absorption or circular dichroism detected become. About that in addition, the binding can be effected by electrical signals, e.g. a capacity measurement be made visible. Furthermore, the binding of endotoxin to the bacteriophage tail proteins also indirectly through displacement experiments be detected.

For the detection according to the invention can the bacteriophage tail proteins if necessary, a separation of the Bacteriophage tail protein-endotoxin complexes from the sample suitable support structures, e.g. Magnetic particles, agarose particles, microtiter plates, filter materials or flow cell chambers, be coupled (indirect evidence). The support structures can e.g. made of polystyrene, polypropylene, polycarbonate, PMMA, cellulose acetate, Nitrocellulose, glass, silicon or agarose exist. The coupling can e.g. can be achieved by adsorption or covalent bonding.

For the depletion process according to the invention the bacteriophage tail proteins are coupled to solid supports. The solid support can Materials for chromatography columns (e.g. Sepharose materials), filtration media, glass particles, magnetic particles, Centrifugation or sedimentation materials (e.g. agarose particles) his.

A functional one is important here Coupling, i.e. Despite that, bacteriophage tail proteins have Binding to the carrier material via for endotoxin accessible Structures. The coupling of the bacteriophage tail proteins can non-specific, or preferably directed, e.g. selective biotinylation, or coupled via a spacer or linker.

The bacteriophage tail proteins can do this with low molecular substances e.g. Be linked to biotin, um over these low molecular weight substances on polypeptides e.g. streptavidin to bind, which in turn were immobilized on the carrier. Instead of The so-called strep tag (Skerra, A. & Schmidt, T.G. M. Biomolecular Engineering 16 (1999), 79-86) can be used, the a short amino acid sequence and binds to streptavidin. His day can also be used be the one about divalent ions (zinc or nickel) or one specific for it antibody (Qiagen GmbH, Hilden) to a carrier material can bind. The strep tag as well as the his tag is preferably used via recombinant DNA technology the recombinantly produced bacteriophage proteins are bound. This Coupling can be directed, e.g. at the N or C terminus or undirected respectively. The directional coupling takes place naturally via a suitable, reactive not common with phage proteins surface-exposed amino acid like cysteine, which has been introduced at a suitable point. Since phage tail proteins are synthesized in the cytoplasm, is not with disulfide bridges to count. Preferably, other amino acids can also be used directly, or as with cysteine above a "spacer" or "CrossLinker" (monofunctional or bifunctional) can be indirectly coupled.

Everyone is in cysteine coupling bifunctional crosslinker with NH and SH reactive groups, with and without intermediate spacer, e.g. 11-Maleimidoundecanoic acid sulfo-NHS or succinimidyl-4- [N-maleimidomethyl] cyclohexane-1-carboxy- [6-amido] caproate possible. If there are no spacers, 8–12 C-atom spacers with a terminal NH group can be used added become. The cysteine coupling is preferably carried out via a specific biotinylation of the cysteine by e.g. EZ-Link PEO-maleimide activated biotin (Pierce).

Divalent ions such as Ca ²⁺ or Mg ²⁺ are important for binding endotoxins to phage proteins such as p12. By adding suitable chelators, such as EDTA or EGTA, this bond can be released. Ca ²⁺ concentrations in the range from approximately 0.1 μM to approximately 100 mM are preferred for binding, particularly preferably in the range from approximately 0.1 μM to approximately 10 mM, particularly preferably in the range from approximately 0.1 μM to approximately 1 mM and furthermore particularly preferably in the range from approximately 10 μM to 1 mM. If the concentration of divalent ions is reduced to below 100 nM by adding 1 mM EDTA, the binding of endotoxin to p12 is released. Mg ²⁺ concentrations above 10 mM impair the binding of endotoxin to p12, which is reflected in an increase in the dissociation constant. Without addition of Mg ²⁺ , a K _d value of 50 nM results and a K _d value of 1 μM was measured in a buffer with 10 mM Mg ²⁺ . Zinc showed an even more inhibitory effect. 1 mM Zn increases the K _d value to 10 μM. Adjustment of the concentration of divalent or other ions (eg: Cu ²⁺ , Al ³⁺ , Zn ²⁺ , Fe ²⁺ , Ca ²⁺ , Ba ²⁺ , Mg ²⁺ , Cd ²⁺ ) to an optimal range for the binding can by substances such as HEDTA, NTA or generally chelators / buffers (ADA: N- [2-acetamido] -2-iminodiacetic acid; 5-AMP: adenosine 5'-monophosphate; ADP: adenosine 5'-diphosphate; ATP: adenosine 5'-triphosphate; Bapta: 1,2-bis (2-aminophenoxy) ethane-N, N, N ', N'-tetraacetic acid; citrate: citric acid; EDTA: ethylenediaminetetraacetic acid; EGTA: ethleneglycol-bis (β-aminoethyl ether) N, N, N ', N'-tetraacetic acid; HEDTA: N-hydroxyethylethylenediaminetriacetic acid; NTA: Nitrilotiracetic acid; SO _4th sulfate), which can be used as a buffer for divalent ions.

The methods according to the invention can therefore further comprise washing steps. Depending on whether a direct or indirect detection or depletion of a separation of sample and bacteriophage tail protein may require washing steps to be built in. Because Ca2 + or other metal ions (e.g. Mg2 +) are essential for the Binding, the binding of endotoxin to e.g. p12 by suitable Washing steps solved become. Depending on the target, whether endotoxin is on the bacteriophage tail protein, e.g. p12 should remain bound, is washed with EDTA-free buffer, when the bond is released should be with EDTA-containing buffer, the EDTA concentrations in the range of at least 0.05 mM to more than 10 mM, preferably is in the range of 2 mM to 5 mM.

The separation takes place after incubation the sample with the corresponding with bacteriophage tail proteins coupled carrier material for about 5-60 min or about 30-180 min or over if necessary Night. For this, the sample is e.g. eluted from the chromatography column, or filtered, or the corresponding particles are centrifuged off or sedimented, or magnetically by applying a magnetic field separated. The separation in the batch process described here, i.e. with sample pre-incubation and with the corresponding bacteriophage tail proteins coupled carrier materials, can be particularly useful at very low endotoxin concentrations his.

The depletion of endotoxins via chromatography columns can but also in the pure flow method. The sample can on the pillar are applied, which is a carrier material with attached bacteriophage tail proteins. The Flow rate is dependent of volume and geometry of the column. The flow rate is also dependent of the volume and endotoxin content of the sample in order to be long contact time between pillar and endotoxin is efficient even at low endotoxin concentrations To achieve depletion. The contact time is the time that the sample is required from application to the column until it flows out.

The separation step can be used, for example, in the depletion process for the regeneration of the bacteriophage tail proteins which are coupled to the solid support. This allows the solid support, For example, a matrix can be reused in a chromatography column. The regeneration is carried out by removing the bound endotoxin through a suitable regeneration buffer containing EDTA or an appropriate chelator. With EDTA, a concentration of greater than 2 mM EDTA is preferred, in particular greater than 10 mM EDTA.

Since ionic interactions always through changes the ionic strength can be influenced also increases or lowering other salts in solution, e.g. NaCl or KCl, the Affect binding of endotoxin to the bacteriophage tail proteins.

To bind in the detection process The protein can also be made visible directly or indirectly by molecular biology or changed biochemically to enable the measurement or improve. To bind endotoxin e.g. directly to p12 Molecular biological exchange of Tyrosine residues can be carried out against tryptophan. For a reduction of the signal background it may be necessary to include the originally contained Exchange tryptophans for tyrosines. To also in protein solutions to be able to measure can p12 after tryptophan introduction additionally chemical be modified. Tryptophan residues are removed by Koshland reagent (2-hydroxy-5-nitrobenzyl bromide) changed in terms of their spectroscopic properties. at displacement experiments can marked, e.g. fluorescence-labeled endotoxin (e.g. Sigma) by endotoxin in the sample e.g. displaced by p12 and determines the concentration of free fluorescent endotoxin become.

With the method according to the invention can detect and remove endotoxin from and in all aqueous solutions become. These solutions can: Proteins, plasmid DNA, genomic DNA, RNA, protein-nucleic acid complexes such as. Phages or viruses, saccharides, vaccines, medicines, Dialysis buffer (medicine), salts or others through endotoxin binding contain contaminated substances.

Another aspect of the invention are bacteriophage proteins to which so-called tags, e.g. the strep or the His tag, preferably at the N or C terminus of the protein, are particularly preferably coupled to the C-terminus. Prefers is the coupling or link of the tags with the bacteriophage proteins via recombinant DNA technology. Production of the nucleic acid, comprising the sequence of the bacteriophage protein and the tag and the production of the expression product is state of the art and need not be explained separately here. Another Aspect of the invention is the nucleic acid sequence, which is a bacteriophage protein encoded along with the Strep or His tag. A particularly preferred one bacteriophage protein modified with the Strep or His tag is the p12 protein from phage T4, however all other bacteriophage proteins are the involved in or responsible for the detection and binding of bacteria are also preferred.

Another aspect of the invention are bacteriophage proteins, with a tag that is a surface exposed Cysteine for specific directed biotinylation, e.g. the tags according to SEQ ID NO: 5, 6 and 7. An example of a p12 with tag is the amino acid sequence listed in SEQ ID NO: 8. A is preferred p12 with one day, especially with a day with a surface exposed Cysteine, in particular a p12 with the tag according to SEQ ID NO: 6 and 7. This Directed biotinylation can also be carried out by a suitable Spacers or linkers can be taught. Furthermore, the present concerns Invention the amino acids with a sequence according to SEQ ID NO: 5, 6 and 7. Furthermore, the present invention relates to coding nucleic acids the amino acid sequence according to SEQ ID NO: 5, 6 and 7.

The methods according to the invention offer compared to Detection and purification procedures for and from endotoxin benefits in the performance of corresponding applications. Furthermore, the manufacture of antibody against LPS cardiac oligosaccharides very difficult, which is corresponding Antibody-based process very much expensive.

The following examples illustrate the Invention and are not to be construed as limiting. Unless otherwise stated, standard molecular biological methods were used, such as. by Sambrook et al., 1989, Molecular cloning: A Laboratory Manual 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

1. Glass jars, plastic jars and buffers

For the endotoxin removal, all glass vessels were de-pyrogenized by baking at 200 ° C (4h) and exclusively pyrogen-free plastic materials (e.g. pipette tips, microtiter plates) used. Other, non-heat-resistant devices or vessels were either Treated with hydrogen peroxide or washed with 1% sodium deoxoycholate. Subsequently it was rinsed with endotoxin-free water. The buffers were largely out endotoxin-free buffer substances (Sigma) and with endotoxin-free water stated. Salts, e.g. NaCl, which can be heated to 200 ° C, were baked (200 ° C, 4h). For chromatographic Buffers used for cleaning were degassed and filtered.

2. Endotoxin detection using LAL test

Endotoxin control evidence was provided with a chromogenic LAL test (Limulus-Amebocyte-Lysate Test, Charles-River Endosafe, Charleston, USA) according to the manufacturer's instructions carried out. Endotoxin standards (Charles-River Endosafe, Charleston, USA) in the range of 0.005–50, or 0.02-50 EU / ml used. The absorption measurement at 405 nm was carried out in a temperature-controlled microtiter plate reader (Genios, Tecan GmbH).

3. Western blot for p12 detection

The detection of p12 in the supernatant of samples treated with beads or in the fractions of affinity chromatography was done by Western blots. Some of the proteins were previously by NaDOC / TCA precipitation (Sodium deoxycholate / tetrachloroacetate) concentrated. Samples were electrophoresed on 12% SDS gels and transferred to PVDF membranes (Immobilon, Millipore). The membranes were washed with PBS for 30 min, blocked with 5% milk powder (1 h) and then with polyclonal anti-p12 antibody incubated (1 h, dilution: 1: 1000). After incubation with, with alkaline phosphatase conjugated secondary antibody (goat anti rabbit IgG) the samples were developed with BCIP / NBT (5-bromo-4-chloroindolylphosphate / nitroblue tetrazolium salt).

4. Endotoxin purification

The cleaning of endotoxin has been done according to the instructions of Galanos, C., Lüderitz, O. & Westphal, O. 1969, Europ. J. Biochem. 9, 245-249 carried out.

Example 5: Specific Coupling of p12 to immobilized iodoacetyl residues:

For directional binding of p12 to the surface the amino acid was reached Serine at position 3 of the strep tag according to SEQ ID NO: 5 by cysteine as in Example 12 and replaced the protein over iodoacetyl residues, which is preferred bind free sulfhydryl residues, immobilized. The resulting p12 was named p12S3C.

There was a 1 ml Sulfolink coupling Poured gel (Pierce), washed with 6 ml of 1% sodium deoxycholate and with 6 ml coupling buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 8.5) equilibrated. Then were 1 ml p12S3C (= N-StrepS3Cp12) (1-1.5 mg / ml in coupling buffer), the column was shaken gently for 15 min, others 30 min without shaking incubated at room temperature, and injected again with 1 ml of p12S3C and the incubation steps repeated. This coupling of p12S3C was Repeated a total of 4 times, and then the column with 6 ml of coupling buffer washed. The runs were collected and the respective p12S3C concentration by absorption measurement determined at 280 nm. 2.2-2.8 mg p12S3C per ml of gel were bound. Subsequently became excess iodoacetyl residues by incubation (45 min) with 1 ml cysteine (50 mM in 50 mM Tris, 5 mM EDTA, pH 8.5) blocked. After washing the column with 16 ml of 1M NaCl and 16 ml of 20 mM Hepes, 150 mM NaCl pH 7.5, the column was ready for use.

The ability of this gel to remove endotoxin from protein solutions was tested with BSA (2-4 mg / ml), carbon anhydrase (1-2 mg / ml) and lysozyme (3-4 mg / ml). BSA and lysozyme solutions were spiked with endotoxin from E. coli O55: B5 (Charles-River Endosafe, Charleston, USA) or E. coli HMS 174 (100-1000 EU / ml), while the carbon anhydrase was not treated with additional endotoxin , In each case 0.5 ml of protein solution was added to the column, incubated for 1 hour at room temperature and then the column was washed with buffer. The proteins were collected in fractions and the endotoxin content before and after the column was determined using a chromogenic LAL test (Charles-River Endosafe, Charleston, USA). Protein recovery was also determined by absorbance measurements at 280 nm. The endotoxins could be removed almost completely (93–99%) from all 3 protein solutions, as in 2A shown. In addition, the proteins were largely eluted from the column (80–99%, 2 B ). The column was then regenerated with 5 mM EDTA, 20 mM Hepes, 150 mM NaCl, pH 7.5. In order to rule out contamination of the protein fractions after the passage over the column by detaching p12, the fractions were examined for p12 using the Western blot technique. No p12 could be detected in the fractions.

Example 6: Unspecific Coupling p12 to NHS-activated carrier material:

N-hydroxysuccinimide (NHS) is displaced from compounds by primary amino residues and is therefore used to couple proteins to surfaces. NHS-activated Sepharose columns (HiTrap NHS-activated HP, 1 ml, Amersham-Pharmacia-Biotech) were first washed with 6 ml of ice-cold 1 mM hydrochloric acid. On Finally, 10-15 ml p12S3C (1.0-3.5 mg / ml) in 0.2 M NaHCO ₃ , 0.5 M NaCl, pH 8.3 were pumped circularly over the column at a room temperature (flow rate 0.8 ml / min). After 60 min the run was collected fractionally and the column washed with 6 ml buffer. The NHS was separated from these fractions by desalting the solution using HiTrap-Desalting columns (5 ml, Amersham-Pharmacia-Biotech) and then the amount of p12 was determined by absorption measurement at 280 nm. 20-25 mg p12S3C were bound to the column. After coupling, the column was repeatedly rinsed in accordance with the manufacturer's instructions with 6 ml of blocking buffer (0.5 M ethanolamine, 0.5 M NaCl, pH 8.3) and washing buffer (0.1 M acetate, 0.5 M NaCl, pH 4.0). The column was then equilibrated with 6 ml of use buffer (20 mM Hepes, 150 mM NaCl, pH 7.5 or 20 mM Tris, 150 mM NaCl, pH 8.5).

Endotoxin removal via this column was tested with lysozyme solutions (3-4 mg / ml in 20 mM Hepes, 150 mM NaCl, pH 7.5 or 20 mM Tris, 150 mM NaCl, pH 8.5). The lysozyme solutions were spiked with E. coli HMS 174 endotoxin (500 EU / ml). 0.5 ml of protein solution was added to the column, incubated for 1 hour at room temperature and then the column was washed with buffer. The lysozyme was collected fractionally and the endotoxin content before and after the column was determined using a chromogenic LAL test (Charles-River Endosafe, Charleston, USA). Protein recovery was also determined by absorbance measurements at 280 nm. 85-90% of the endotoxins were removed from the solution as in 3A and 85–90% of the lysozyme could be eluted from the column again by washing with use buffer ( 3B ). The column was then washed with 6 ml of 5 mM EDTA, 20 mM Hepes, 150 mM NaCl, pH 7.5 and 6 ml of 1 M NaCl. In order to rule out contamination of the protein fractions after the passage over the column by detaching p12, the fractions were examined for p12 using the Western blot technique. No p12 could be detected in the fractions.

Example 7: Directed Coupling from p12 to via Diaminoethane and N-succinimidyl iodoacetate (SIA) as spacers on NHS-activated Support material column.

To be bound to that Chromatography support material to achieve a bifunctional linker on NHS-activated surface bound by a coupling of p12S3C via its free cysteine and Iodoacetyl residues of the bifunctional linker enables.

NHS-activated Sepharose columns (HiTrap NHS-activated HP, 1 ml, Amersham-Pharmacia-Biotech) were first washed with 6 ml of ice-cold 1 mM hydrochloric acid, then 1 ml of ethylenediamine (10 mg / ml in 0.2 M NaHCO ₃ , 0.5 M NaCl , pH 8.3) and the column was incubated for 30 min at room temperature. After blocking excess NHS groups with ethanolamine (0.5 M ethanolamine, 0.5 M NaCl, pH 8.3) and washing (0.1 M acetate, 0.5 M NaCl, pH 4.0) the column was washed with 6 ml borate buffer (50 mM sodium borate, 150 mM NaCl, 5 mM EDTA, pH 8.3). Subsequently, 10 ml of N-succinimidyl iodoacetate (SIA, Pierce, 200 μl of SIA stock solution in 10 ml of borate buffer; SIA stock solution: 1.4 mg of SIA in 1 ml of DMSO) were circularly rinsed over the column for 30 min. The column was then washed with 6 ml borate buffer and p12S3C (1 mg / ml, 50 ml in borate buffer) rinsed over the column for 1 hour. Excess iodoacetyl residues were saturated with 1 ml cysteine solution (5 mM cysteine in borate buffer, incubate for 15 min at room temperature) before the column with the use buffers (20 mM Hepes, 150 mM NaCl, pH 7.5 or 50 mM Tris, 150 mM NaCl, pH 8.5 ) were equilibrated. The coupling reactions with SIA were carried out in the dark.

Endotoxin removal via this column was tested with lysozyme solutions (3-4 mg / ml in 20 mM Hepes, 150 mM NaCl, pH 7.5 or 20 mM Tris, 150 mM NaCl, pH 8.5). The lysozyme solutions were spiked with E. coli HMS 174 endotoxin (500 EU / ml). 0.5 ml of protein solution was added to the column, incubated for 1 hour at room temperature and then the column was washed with buffer. The lysozyme was collected in fraction white and the endotoxin content before and after the column was determined by means of a chromogenic LAL test (Charles-River Endosafe, Charleston, USA). Protein recovery was also determined by absorbance measurements at 280 nm. 90% of the endotoxins were removed from the solution, as in 3A and 75–85% of the lysozyme could be eluted from the column again by washing with use buffer ( 3B ). The column was then washed with 6 ml of 5 mM EDTA, 20 mM Hepes, 150 mM NaCl, pH 7.5 and 6 ml of 1 M NaCl. In order to rule out contamination of the protein fractions after the passage over the column by detaching p12, the fractions were examined for p12 using the Western blot technique. No p12 could be detected in the fractions.

Example 8: Removal of endotoxin from a BSA solution in the flow process

Hi-Trap-NHS activated Sepharose (Amersham Biosciences, Uppsala, Sweden) was non-specifically coupled to p12 via primary amino groups according to the manufacturer's instructions. 8 mg p12 / ml gel material were immobilized covalently. The 1 ml chromatography column thus obtained was equilibrated at a flow rate of 1 ml / min with 10 ml buffer A (20 mM HEPES pH 7.5, 150 mM NaCl, 0.1 mM CaC12). 4 ml of a BSA solution (11.5 mg BSA (Carl Roth GmbH, Germany) / ml buffer A) were then applied (In jection: I) and the run (E) collected in 2.5 ml fractions. The column was then washed with 15 ml of buffer A and the endotoxin bound to the column was eluted with 7 ml of buffer B (20 mM HEPES pH 7.5, 150 mM NaCl, 2 mM EDTA). 2 ml fractions were collected in each case during washing and elution. After each experiment, the column was regenerated with 20 ml buffer C (20 mM HEPES pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.1% sodium deoxycholate). The endotoxin concentration was determined by a chromogenic Limulus Amebocyte Lysate (LAL) test (Charles-River Endosafe, Charleston, USA), according to the manufacturer's instructions. The protein concentration was determined by measuring the UV absorption. The endotoxin removal efficiency was between 95-99% and the protein loss was about 6-10%.

Example 9: Removal low amounts of endotoxin from buffer using non-specifically coupled p12.

20 ml NHS-activated Sepharose 4 FastFlow (Amersham Biosciences) were first washed with ice-cold hydrochloric acid and subsequently with 292 mg p12 (7 mg / ml in 25 mM citrate pH 7.0) with shaking for 4 hours Incubated at room temperature. Then the Sepharose was with 7 × 80 ml of 5 mM citrate pH 2.0 and 1 ml of the wash fractions dialyzed against 5 mM citrate pH 2.0. These dialysates were used for the excess p12 in the wash fractions by means of absorption measurement at 280 nm quantify. There was a loading density of 8.7 mg p12 per 1 ml of Sepharose determined. Unreacted NHS residues were removed Saturated 12 h incubation of Sepharose with 1M Tris pH 8.0. With this pillar material became pillars poured with 2 ml volume and this at 4 ° C in 20% ethanol until use stored.

In 3 parallel experiments, 4 ml of endotoxin solution (S) was applied to each column (see 9 ). The endotoxin solution consisted of endotoxin from E. coli O55: B5 (Charles-River Endosafe, Charleston, USA) in equilibration buffer (20 mM Hepes, 150 mM NaCl, 0.1 mM CaCl ₂ , pH 7.5). The endotoxin concentration of this solution was 4.6 EU / ml.

The pillars were initially with 12 ml regeneration buffer (20 mM Hepes, 150 mM NaCl, 2 mM EDTA, pH 7.5) and then rinsed with 12 ml equilibration buffer. Then was again add equilibration buffer to the column and fractionate 1 ml.

The endotoxin solution was applied to the columns (I) and fractions of 5 ml and 2 ml collected. Then was the pillar regenerated with 4 ml regeneration buffer (B). In the run fractions no endotoxin could be detected, i.e. the endotoxin impurities could be completely removed in all three experiments.

Example 10: non-specific Coupling of biotinylated p12 to magnetic streptavidin beads.

p12 (3 mg / ml in PBS, 0.05% Tween 20) with sulfo-NHS-LC-LC-Biotin (Pierce), in a ratio of 1 : 10 to 1: 20 incubated at RT for one hour and then against Buffer (e.g. PBS or 20 mM Hepes, 150 mM NaCl, 5 mM EDTA, pH 7.5) dialyzed. NHS-activated biotin binds to primary amino residues from p12. Subsequently were made into 1 ml streptavidin beads (MagPrep streptavidin beads, Merck) 50 ul biotinylated p12 (1 mg / ml), shaken at room temperature for 2 hours and then excess p12 by washing four times with 1.5 ml of 20 mM Tris, 10 mM EDTA, pH 7.5 removed.

Endotoxin removal was carried out with buffer (20 mM Hepes, 150 mM NaCl, pH 7.5) and protein solutions (0.1 mg / ml BSA, 0.1 mg / ml lysozyme, 0.1 mg / ml carbon anhydrase in 20 mM Hepes, 150 mM NaCl, pH 7.5) tested. The buffer and the BSA and lysozyme solution were spiked with 5 EU / ml (endotoxin from E. coli O55: B5, Charles-River Endosafe, Charleston, USA). The carbon anhydrase solution contained about 1 EU / ml. 25 μl of magnetic beads with immobilized p12 were added to 200 μl of buffer or protein solution, mixed by pipetting up and down and incubated at room temperature for 30 min. The beads were removed from the solution using a magnet and the supernatant was pipetted off. The endotoxin content of untreated samples and samples incubated with beads was then determined using the LAL test and the protein recovery was determined by measuring the absorption at 280 nm. The endotoxin was almost completely removed from the buffer (99.9% endotoxin removal, 4A ) and also from the protein solution the endotoxin was reduced by 70–92% ( 4B ) depleted. Protein recovery ranged from 57% to 99% (BSA: 87%, carbon anhydrase: 99%, lysozyme: 57%; 4B ).

Example 11: non-specific Coupling of biotinylated p12 to immobilized streptavidin.

P12 (3 mg / ml in PBS, 0.05% Tween 20) was incubated with sulfo-NHS-LC-LC-Biotin (Pierce), in a ratio of 1:10 to 1:20 for one hour at RT and then against buffer (e.g. PBS or 20 mM Hepes, 150 mM NaCl 5 mM EDTA, pH 7.5) dialyzed. NHS-activated biotin binds to primary amino residues of p12. The biotinylated p12 is then incubated for 1 h at room temperature with streptavidin-loaded chromatography material (ImmunoPure immobilized streptavidin: 6% cross-linked agarose beads) and excess siges p12 removed by washing with PBS.

Endotoxin removal was tested with buffer (20 mM Tris, 150 mM NaCl, pH 8.0) and BSA (0.5 mg / ml in 20 mM Tris, 150 mM NaCl, pH 8.0). 1 ml of buffer or BSA solution were spiked with 10 EU / ml, 50 μl of p12 agarose were added and shaken for 1 hour at room temperature. The p12 agarose was then centrifuged off and the endotoxin and protein concentration in the supernatant measured. 99% endotoxin was removed from the buffer and 86% from the BSA solution ( 5 ). 90% of BSA was recovered.

Example 12: Studies on the p12 endotoxin binding by means of surface plasmon resonance measurements

The binding of p12 to endotoxin or to bacteria, via the lipopolysaccharides in the outer cell membrane, was examined by means of surface plasmon resonance measurements (Biacore J). In order to determine the dissociation constant (K _d ), endotoxin from E. coli O55: B5 (Sigma) was immobilized on a hydrophobic HPA chip in accordance with the manufacturer's instructions and p12 was injected in various concentrations ( 6A ). The binding is measured in relative "response units" (RU) and the equilibrium values are plotted against the associated p12 concentrations ( 6B ). The K _d value was determined by adapting the Langmuir adsorption isotherm (RU = (RU _maX · [p12]) / ([p12] + K _d )) to these data (Table 1). Endotoxin-free buffers were used for the measurements. K _d values in the range from 10 ⁻⁷ to 10 ⁻⁹ M were determined for pH values between 6 and 10 (Table 1). Binding was broken by injection of 1 mM or 5 mM EDTA and the chip regenerated.

Tabelle 1: Dissoziationskonstanten von Endotoxin an p12 in Abhängigkeit von dem pH-Wert der Lösung.

Table 1: Dissociation constants of endotoxin at p12 depending on the pH of the solution.

In order to investigate the binding of bacteria to p12, biotinylated p12 was immobilized on streptavidin chips and various E. coli strains were injected. The bacteria were recorded in PBS for the measurements. E. coli strains were used which have lipopolysaccharides with different proportions of polysaccharides. The polysaccharide part consists of a "heart" region, which is linked to lipid A and the so-called O-antigen. The O-antigen varies greatly between different types of bacteria and also bacterial strains, while the "heart" region is highly conserved. Strains that have the "heart" region and O-antigen (eg E. coli), as well as strains that have a complete "heart" region (E. coli D21) were bound by p12, while strains with a greatly shortened "heart "Region (e.g. E. coli D21f2) was no longer recognized by p12 ( 6C ). The binding could be released by EDTA (5 mM) and the chip regenerated.

Example 13: recombinant p12 constructs

• 1. Construction of p12 with N-terminal Strep-Tag (N-Strep-p12): By means of PCR, the nucleotide sequence for the Strep-Tag (US patent 5,506,121) was introduced at the 5 'end of the T4p12 gene. For this, a primer was constructed for the 5 'end of the p12 gene (5'-GAA GGA ACT AGT CAT ATG GCT AGC TGG AGC CAC CCG CAG TTC GAA AAA GGC GCC AGT AAT AAT ACA TAT CAA CAC GTT-3' (SEQ ID NO: 1), which contains the nucleotide sequence of the strep tag at its 5 'end (in italics in the sequence) and has a restriction site (NdeI, underlined in the sequence) such that the gene is inserted into the expression plasmid in the correct reading frame A primer was constructed for the 3 'end of the p12 gene, which introduces a BamH I restriction site (italics in the sequence) behind the p12 gene (5'-ACG CGC AAA GCT TGT CGA CGG ATC CTA TCA TTC TTT TAC CTT AAT TAT GTA GTT-3 '), (SEQ ID NO: 2). The PCR was carried out with 40 cycles (1 min 95 ° C, 1 min 45 ° C and 1 min 72 ° C). Approach was cut with the restriction endonucleases NdeI and BamHI and the desired fragment according to size fraction tion via an agarose gel and elution from the gel into the NdeI and BamHI site of the expression plasmid pET21a. The sequence of the N-Strep-pl2 gene was checked for correctness by DNA sequencing. The further steps to the plasmid pNS-T4p12p57 were carried out as described by Burda, MR & Miller, S. (Eur J Biochem. 1999 265 (2), 771-778) for T4p12p57. The plasmid pNS-T4p12p57 was then transformed into the expression strain BL21 (DE3).
• 2. Paste an N-terminal cysteine residue in N-Strep-p12 (N-Strep-S3C-p12 and N-Strep-S14C-p12): The insertion an N-terminal cysteine residue was described as under 1. carried out, being for that two new primers for constructed the 5 'end were. For the N-Strep-S3C-p12 became the primer 5'-GAA GGA ACT AGT CAT ATG GCT TGT TGG AGC CAC CCG CAG TTC GAA AAA GGC GCC AGT AAT AAT ACA TAT CAA CAC GTT-3 '(SEQ ID NO: 3), for the N-Strep-Sl4C-p12 became the primer 5'-GAA GGA ACT AGT CAT ATG GCT AGC TGG AGC CAC CCG CAG TTC GAA AAA GGC GCC TGT AAT AAT ACA TAT CAA CAC GTT-3 '(SEQ ID NO: 4) is used.
• 3. Purification of N-Strep-p12 Protein: The E. coli strain BL21 (DE3) with the plasmid pNS-T4p12p57 in 2 l shake cultures (LB medium with Ampicillin 100 μg / ml) up to an OD600 of 0.5-0.7 at 37 ° C was drawn and the expression of the N-Strep-pl2 protein was determined by Add 1 mM IPTG (isopropyl-β-thio-galactopyranoside) induced. After incubation at 37 ° C for 4h the cells were harvested. Harvested cells from 10 L culture were taken up in 50 ml sodium phosphate, 20 mM pH 7.2, 2 mM MgSO4, 0.1 M NaCl, broken up by three French press treatments (20,000 psi) and subsequently Centrifuged at 15,000 rpm (SS34) for 30 min. After washing twice in the same buffer the N-Strep-p12 protein from the pellet the pellet 3x with stirring for 30 min in 40 mM TrisHCl pH 8.0, 10 mM EDTA extracted, the mixture for 30 min Centrifuged at 15,000 rpm (SS34) and the detached NS-p12 in the supernatant at 4 ° C stored. The extraction was repeated twice. and the combined supernatants were on a streptactin affinity column (15 ml), equilibrated with buffer "W" (100 mM TrisHCl pH 8, 1 mM EDTA, 150 mM NaCl), applied (IBA GmbH, Göttingen). After washing with 5 column volumes Buffer "W" was with 3 volumes Buffer "W" with 2.5 mM desthiobiotin in buffer "W" eluted. After repeated Dialysis against buffer "W" and concentration was carried out via SDS-PAGE and UV spectroscopy (Burda et. al. 1999) the concentration and Purity of N-Strep-T4p12 determined. 10 liters of culture became about 100 mg N-Strep-T4p12 cleaned.

SEQUENCE LISTING

Claims (15)

A method for the detection of endotoxin comprising the steps: a) incubating a sample with a bacteriophage tail protein, b) Detection of endotoxin bound to bacteriophage tail proteins. The method of claim 1, optionally further comprehensive after step a) and before step b) the additional step a ') Separation of the bacteriophage tail protein-endotoxin complexes from the sample. Method according to one of claims 1 to 3, wherein the detection is carried out by means of spectroscopic methods. Process for removing endotoxin from a Sample, comprising the steps: a) incubation or in contact bring a sample of bacteriophage tail proteins that are non-specific or directed, immobilized on a solid support, b) Separate the bacteriophage tail protein-endotoxin complex from the sample. The method of claim 4, wherein steps a) and b) in a chromatography column flow process carried out become. The method of claim 4, wherein the solid support is filtration media, Glass particles, magnetic particles, centrifugation, sedimentation materials or filling materials for chromatography columns. The method of claims 4 to 6, wherein the bacteriophage tail proteins are linked via coupling groups the solid support are immobilized. The method of claim 7, wherein the coupling group is a lectin, receptor or anticalin. The method of claim 7, wherein the coupling group is a streptavidin or avidin and the bacteriophage tail proteins are coupled with biotin or a strep tag. The method of claims 4 to 6, wherein the bacteriophage tail proteins covalently over chemical bonds are immobilized on the solid support. Method according to one of the preceding claims, wherein the bacteriophage tail protein is a strep tag or a his tag having. The method of claim 11, wherein the tag is an amino acid sequence according to SEQ ID NO. 5, 6 or 7. The method of claim 11 or 12, wherein as a bacteriophage tail protein the p12 protein of the phage T4 is used. Method according to one of the preceding claims, wherein the Ca ²⁺ concentration in the incubation is 0.1 μM to 10 mM and the Mg ²⁺ concentration is 0.1 μM to 10 mM. Method according to one of claims 1 to 3, wherein marked Endotoxin from binding to a bacteriophage tail protein repressed and the labeled endotoxin is subsequently detected.