Uses, methods, and products related to lipopolysaccharide-bound oligomeric proteins

Abstract

The present invention provides and describes the use of an oligomeric protein as a binding agent for binding to lipopolysaccharide (LPS), the oligomeric protein having a coiled-coil structure comprising at least two monomeric peptides, each of which may be the same or different, is capable of forming an α-helix, and comprises at least one core sequence having at least 60% sequence identity with the heptad (7 amino acid) repeat sequence of SEQ ID NO: 1. Also provided and described herein are methods for binding to, detecting, and removing LPS, and products comprising the oligomeric protein.

Description

[Technical Field]

[0001] This specification describes the use of an oligomeric protein having a coiled-coil structure as a binder for binding to lipopolysaccharide (LPS). This specification also describes methods for binding to LPS, methods for detecting LPS, methods for removing LPS, and products containing this oligomeric protein. [Background technology]

[0002] Lipopolysaccharide, also known as endotoxin (in this specification, the terms "LPS" and "endotoxin" are used interchangeably), is an essential component of the outer membrane of Gram-negative bacteria. Lipopolysaccharide consists of three structural components: lipid A; core oligosaccharide; and O antigen. Lipid A contains two phosphoglucosamine molecules, each having four acyl chains linked by oxygen and two acyl chains linked by nitrogen. These acyl chains are embedded in the bacterial outer membrane, thus anchoring the LPS within the bacterial membrane. Core oligosaccharide (COS) is a non-repeating structure composed of various sugars, linked to lipid A by glycosidic bonds. Finally, the O antigen is a polymer composed of 4-40 repeating tetrasaccharide monomers, with an average of 30 repeats (Non-Patent Literature 1). The O antigen is linked to the second-to-last sugar of COS at the end opposite lipid A, although some forms of LPS lack the O antigen. LPS lacking the O antigen is conventionally called "rough" LPS in contrast to wild-type "smooth" LPS (Non-Patent Literature 2).

[0003] The lipid A component of LPS and the sugar most proximal to it are highly conserved across Gram-negative bacterial species, while the rest of the core oligosaccharide and the O antigen are significantly less conserved and can vary between bacterial species and serotypes (Non-Patent Literature 3).

[0004] Endotoxins are highly toxic to animals, particularly humans, due to their lipid A tendency to activate Toll-like receptor 4, and thus can induce extreme immune responses that can cause sepsis and toxic shock at low doses of 1 μg per kg of body weight. Due to the ubiquity of Gram-negative bacteria in the biological environment, endotoxins are common impurities in the manufacture of drugs, vaccines, and laboratory equipment and reagents. Given the potential health effects associated with endotoxin contamination, it is important to remove any endotoxins that may be present in products intended for human consumption as much as possible before use.

[0005] Currently, various methods and products are available for endotoxin removal. For laboratory use, these methods and products include spin column filters or flow columns packed with resins linked to one or more endotoxin-binding molecules. Samples can be applied to these filters or columns, and the endotoxin-binding molecules bind to any endotoxins present, thus removing them from the sample. Known endotoxin-binding molecules that can be used in these products include the lipid A-binding antibiotic polymyxin B and polylysine polymers that bind to endotoxins by electrostatic interactions. However, these endotoxin-binding molecules currently available have limitations. The reported binding affinity of polymyxin B is only in the micromolar concentration range depending on the strain in question, and is therefore unsuitable for binding to and removing low concentrations of endotoxins from samples. On the other hand, polylysine has a strong positive charge and therefore interacts nonspecifically with the negatively charged phosphate groups of lipid A and core oligosaccharides. Therefore, this mechanism of action is unsuitable for use at all pH values ​​or with all LPS types. Furthermore, polylysine may interact with and thus remove other negatively charged molecules that may be present in the sample.

[0006] Other methods include ion exchange chromatography. Ion exchange chromatography is commonly used in the pharmaceutical industry for the purification of pharmaceutical products and typically relies on electrostatic interactions between negatively charged LPS and positively charged immobilized ligands. However, using ion exchange chromatography to remove endotoxins from strongly charged samples can cause problems. For example, if the sample contains strongly positively charged particles, these particles will compete with the immobilized ligands for capturing LPS. Conversely, if the sample contains strongly negatively charged particles (not LPS), these particles will compete with LPS for binding to the immobilized ligands. In both cases, these undesirable reactions reduce the efficiency of LPS removal. The electrostatic interactions that underlie ion exchange chromatography can be disrupted even in samples with high ionic strength, and therefore these methods are not suitable for all scenarios.

[0007] In addition to removing endotoxins from samples, it is also desirable to be able to detect endotoxins, thereby ensuring that therapeutic products, devices, reagents, etc., are "endotoxin-free" and therefore safe for therapeutic use. In this regard, the most common method for detecting endotoxins is currently the Lumulus amebocyte lysate (LAL) assay. The LAL assay is approved by the FDA and EFSA for the detection of endotoxins in medical and therapeutic products, with a detection range of 1 picomolar concentration (0.1 EU / mL). This assay uses a lysate of amebocytes found in the blood of horseshoe crabs (Limulus), which contains a complex mixture of proteins and enzymes. More specifically, the LAL assay is based on the activity of the enzyme "(horseshoe crab coagulation) factor C" (commonly known simply as factor C), which is activated upon LPS binding. Factor C is a trypsin-type serine protease that activates a complex cascade of downstream enzymatic reactions, ultimately indicating the presence of LPS. However, this reaction cascade can also be activated by β-glucans, which are commonly found in a range of bacteria, fungi, and plants. Therefore, β-glucans can cause false-positive results in LAL assays. Furthermore, amebosite lysates are very expensive to produce, and current production methods are not sustainable.

[0008] Overfishing of horseshoe crabs has necessitated the development of alternative methods for endotoxin detection. A similar assay was developed using recombinantly expressed C factor to cleave the pigment-producing substrate, thus enabling more direct detection of LPS. However, due to its complex composition, it remains expensive.

[0009] Furthermore, the activity of factor C can be easily disrupted by factors such as temperature or pH fluctuations, denaturing compounds like organic solvents, urea, or strong surfactants, and common protease inhibitors. Batch-to-batch variability can also occur between different factor C preparations. This makes the enzyme difficult to use and means that results obtained using factor C-dependent LAL assays are often inconsistent or unreproducible.

[0010] A further challenge in LPS detection, affecting all detection methods, is the tendency of LPS to aggregate. It is well known in this field that endotoxin molecules tend to form aggregates in aqueous solutions. This aggregation is due to cations (especially Ca) in the solution. 2+ and Mg 2+ Aggregation is increased by the presence of divalent cations (such as ions) and surfactants that can form micelles around LPS. This aggregation reduces the amount of measurable LPS in the solution and thus hinders the detection of low concentrations of LPS. This effect is known as "LPS masking" and can be caused by a wide range of different agents. For example, there are several compounds in blood samples that can mask LPS, such as LPS-binding proteins, anti-LPS antibodies, and divalent cations. Furthermore, endotoxin molecules from different bacterial sources may have different molecular weights and exhibit different aggregation behaviors, which can result in variable results when measuring the same concentration of LPS from different sources. Therefore, it may be useful to provide improved LPS binders that can be used to remove or detect LPS. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Peterson, AA and McGroarty, EJ, "High-molecular-weight components in lipopolysaccharides of Salmonella typhimurium, Salmonella minnesota, and Escherichia coli," Journal of Bacteriology, vol. 162, pp. 738-745 (1985). [Non-Patent Document 2] Hitchcock, PJ et al., "Lipopolysaccharide nomenclature — past, present, and future," Journal of Bacteriology, doi:10.1128 / jb.166.3.699-705.1986. [Non-Patent Document 3] Bertani, B. and Ruiz, N., "Function and Biogenesis of Lipopolysaccharides," EcoSal Plus, Vol. 8, No. 1, doi:10.1128 / ecosalplus.esp-0001-2018. [Overview of the project]

[0012] The inventors have developed a novel LPS-binding agent in the form of an oligomeric protein having an α-helix coiled-coil structure.

[0013] The novel LPS-binding agents disclosed herein are based on an α-helix coiled-coil structure that can be found in the yeast transcription factor GCN4, in which a short C-terminal section (stretch) of this protein forms a highly stable dimeric coiled-coil structure called a leucine zipper.

[0014] Therefore, in the first aspect, the use of an oligomeric protein as a binder for binding to lipopolysaccharide (LPS) is provided herein, the oligomeric protein having a coiled-coil structure and comprising at least two monomer peptides, each monomer peptide may be the same or different, and capable of forming an α-helix, and comprising at least one core sequence having at least 60% sequence identity with the heptad (7 amino acid) repeat sequence of SEQ ID NO: 1.

[0015] Like coiled-coil proteins, which contain or are composed of amphiphilic α-helices (or α-helices chains), coiled-coil oligomeric proteins have a hydrophobic core. The hydrophobic core contains hydrophobic residues that face each other within the hydrophobic core structure.

[0016] Therefore, more specifically, the core sequence of the peptide monomer may contain at least three heptad motifs abcdefg or variants thereof, each variant containing one or fewer insertions or deletions to the heptad motif. Furthermore, in one embodiment, at least 50% of the amino acid residues corresponding to positions a and d of the heptad motif or variant are hydrophobic residues. In another embodiment, at least 75% of the amino acid residues corresponding to positions a and d of the heptad motif or variant are hydrophobic residues.

[0017] Therefore, in one embodiment of this aspect, the use of an oligomeric protein as a binder for binding to lipopolysaccharide (LPS) is provided herein, the oligomeric protein having a coiled-coil structure and comprising at least two monomeric peptides, each monomeric peptide may be the same or different and capable of forming an α-helix, and comprising at least one core sequence having at least 60% sequence identity with the heptad repeat sequence of SEQ ID NO: 1, the core sequence comprising at least three heptad motifs abcdefg or variants thereof, each variant comprising one or fewer insertions or deletions into the heptad motif, and at least 50% of the amino acid residues corresponding to positions a and d of the heptad motif or variants thereof are hydrophobic residues.

[0018] The amino acid residue composition of the hydrophobic core of a coiled-coil protein does not have to consist entirely of hydrophobic residues or only hydrophobic residues; other residues, including hydrophilic residues such as polar residues, may be present. Accordingly, in certain embodiments, at least 52.5, 55, 60, 62.5, 70, or 75% of the amino acid residues corresponding to positions a and d of the heptad motif or its variants are hydrophobic residues.

[0019] In a second aspect, the Specified herein provides a method for binding to LPS, the method comprising the step of contacting LPS or a sample containing LPS with an oligomeric protein as defined herein, thereby enabling the protein to bind to LPS so as to form a protein-lipopolysaccharide complex.

[0020] In one embodiment, the method is an in vitro method.

[0021] In a third aspect, a kit used as a binder for binding to LPS as defined herein, or used in a method for binding to LPS as defined herein, (i) with an oligomeric protein as defined herein; (ii) at least one type of non-ionic surfactant, and A kit containing the same is provided.

[0022] The uses and methods herein may be used in detecting and / or removing LPS in a sample or LPS from a sample.

[0023] Due to its ability to bind a wide range of endotoxins with high affinity, this oligomeric protein is suitable for use in various applications including endotoxin binding, detection and removal. In this regard, this oligomeric protein may be immobilized on a solid substrate. For example, this oligomeric protein is immobilized on a resin for use in a column or filter, such as a spin column filter or a flow column, and binds to and removes endotoxin from a sample applied to the filter or column as in the endotoxin removal method outlined above. This oligomeric protein may also be used in an endotoxin detection system for both detecting the presence of endotoxin in a given sample and ensuring that samples, reagents, products, etc. are endotoxin-free and no endotoxin is detected. In this regard, the oligomeric protein may be provided in the form of a conjugate or fusion with a second component, such as a conjugate with a detection moiety or a fusion protein with a suitable fusion partner, to facilitate the detection of endotoxin.

[0024] In a fourth aspect, herein provided is a product comprising an oligomeric protein as defined herein immobilized on a solid substrate.

[0025] As described above, the oligomeric protein as defined herein is understood to interact with LPS via the lipid A moiety. Thus, in a fifth aspect, herein provided is the use of an oligomeric protein as defined herein as a binder for binding to the lipid A of LPS.

[0026] Similarly, in a sixth aspect, a method for binding LPS to lipid A is provided herein, the method comprising the step of contacting lipid A or a sample containing lipid A with an oligomeric protein as defined herein, thereby enabling the protein to bind to lipid A so as to form a protein-lipopolysaccharide complex.

[0027] In the seventh aspect, the Specified herein provides a kit used as a binder for binding to lipid A as defined herein, or for use in a method for binding to lipid A as defined herein, the kit is, (i) with an oligomeric protein as defined herein; (ii) at least one non-denaturing surfactant, Includes.

[0028] The oligomeric proteins described herein provide alternative binding agents for LPS. In one embodiment, the disclosure herein provides an improved binding agent for LPS.

[0029] The LPS binding agent described herein has several advantages. Furthermore, it can be seen that this binding agent addresses several of the problems outlined above that are associated with known LPS binding and detection methods.

[0030] From the perspective of LPS detection, the oligomeric proteins described herein eliminate the need for expensive lysis solutions extracted from horseshoe crabs and avoid the consistency and reproducibility issues associated with LPS detection methods that rely on the use of factor C, such as the LAL assay and its recombinant variants.

[0031] Furthermore, the oligomeric proteins described herein can dissolve LPS aggregates. Therefore, these oligomeric proteins can reduce LPS masking and effectively increase the measurable concentration of LPS in samples containing such LPS aggregates. This makes it possible to detect low concentrations of LPS in the sample.

[0032] The oligomeric proteins described herein contain relatively short peptide sequences, and therefore, in some embodiments, these oligomeric proteins may be synthesized without requiring any biological expression system.

[0033] Next, the present invention will be described in more detail in the following description and non-limiting embodiments with reference to the following drawings. [Brief explanation of the drawing]

[0034] [Figure 1A] Figure 1A is a side view showing the structure of the GCN4-pII trimer, sourced from PDB-ID 2YO0 (Hartmann et al., 2012). [Figure 1B] Figure 1B is a front view showing the structure of the GCN4-pII trimer, sourced from PDB-ID 2YO0 (Hartmann et al., 2012), with the coisoleucine residues at positions a and d colored green. [Figure 2]Figure 2 shows a schematic representation of the general structure of LPS based on LPS derived from S. typhimurium. The lipid A portion (inset) consists of two phosphoglucosamines, with four oxygen-linked acyl chains and two nitrogen-linked acyl chains embedded in the outer membrane. The core oligosaccharide (COS) is linked to lipid A by a glycosidic bond, with the O antigen linked to the second-to-last COS sugar. The O antigen consists of tetrasaccharide repeats in a number that varies between 4 and 40 repeat units, with an average of 30 repeats (Peterson and McGroarty, 1985). Lipid A and the two proximal 3-deoxy-D-manno-octa-2-urosonic acid (KDO) sugars are highly conserved among Gram-negative species, while the remainder of COS and the O antigen are conserved among bacterial species and serotypes, respectively. [Figure 3] Figures 3A to 3D show SPR binding curves after injecting various LPS components into immobilized K9-GCN4-PII. Vertical lines indicate the start and end of injection. Figure 3A shows the injection of whole LPS. Figure 3B shows the injection of rough LPS lacking the O antigen. Figure 3C shows the injection of deep rough LPS lacking all sugars except two proximal KDOs. Figure 3D shows the injection of LPS polysaccharide lacking lipid A. [Figure 4] Figure 4 shows graphs of the SPR difference values ​​at the end of injection, normalized by the molar concentration of the ligand, for each of the LPS components tested in Figure 3. [Figure 5] Figure 5 shows the ELITA binding curves of LPS to two GCN4-containing constructs, K9-His (left) and K14-His (right). [Figure 6] Figure 6 shows TEM images of LPS alone (top) and LPS with GCN4-pII (bottom) at 4k magnification (left) and 8k magnification (right). [Figure 7]Figure 7 shows a schematic overview of the produced constructs. Constructs derived from SadA were first described by Alvarez et al. (Alvarez et al., 2008) and Hartman et al. (Hartman et al., 2012). The andreinlvpas construct was first described by Deiss et al. (Deiss et al., 2014). The GCN4 construct was synthesized by GenScript (GenScript Biotech Corp). [Figure 8] Figures 8A to 8D show the SPR Fc1, Fc2, and Fc1-F2 curves for K9 immobilized with various S. tiphimulium LPS components. Figure 8A shows the case of smooth LPS. Figure 8B shows the case of rough LPS. Figure 8C shows the case of deep rough LPS. Figure 8D shows the case of polysaccharides derived from LPS. [Figure 9] Figures 9A to 9D show the SPR Fc1, Fc2, and Fc1-F2 curves for K14 immobilized with various S. tiphimurium LPS components. Figure 9A is for smooth LPS. Figure 9B is for rough LPS. Figure 9C is for deep rough LPS. Figure 9D is for polysaccharides derived from LPS. [Figure 10] Figures 10A to 10D show SPR Fc1, Fc2, and Fc1-F2 comparison curves for immobilized K3 (Fc1 channel) and K3-His (Fc2 channel) with various S. tiphimurium LPS components. Figure 10A is for smooth LPS. Figure 10B is for rough LPS. Figure 10C is for deep rough LPS. Figure 10D is for polysaccharides derived from LPS. [Figure 11] Figure 11 shows the absolute values ​​(top) and composition and negative control (bottom) of ELITA experiments using K9 and K14. SadA = Salmonella component K9 or K14. BSA = Bovine serum albumin. TSP = Phage tail spike protein. ST-HRP = Horseradish peroxidase-conjugated streptoactin. [Figure 12] Figure 12 shows the CD spectra of GCN4-pII alone and in the presence of LPS. It can be seen that there is minimal change in the secondary structural composition of GCN4-pII before and after binding to LPS. [Figure 13] Figure 13 shows a graph of the results of a LAL assay demonstrating the masking effect of GCN4-pII at concentrations ranging from 10 to 0.1 μM, spiked with 0.5 EU / mL LPS. Optimal masking was observed at 1 μM GCN4-pII, with a masking effect of 89% of the total signal. [Figure 14] Figure 14 shows the fingerprint region of the 2D 1H-1H tocsy NMR spectrum of GCN4-pII. All 29 predicted spin systems were well resolved, assignable without any signs of peak splitting, and the sample was homogeneous in solution. [Figure 15] Figure 15 shows a graph of the results of a GCN4-pII-based ELISA using biotinylated LPS for detection. [Figure 16] Figure 16 shows a graph of the results of the LAL assay using the same sample as in the assay in Figure 15. [Figure 17] Figure 17 shows a graph comparing the results of the GCN4-pII-based ELISA and the LAL assay. [Figure 18] Figure 18 shows SPR binding curves for various LPS types and PBS-E as a control running buffer. [Figure 19] Figure 19 shows the phylogenetic distribution of the LPS mutant used in Example 5. This figure is from Bern and Goldberg, 2005. [Modes for carrying out the invention]

[0035] explanation The oligomeric proteins disclosed herein have an α-helix coiled-coil oligomeric structure. A coiled-coil is a ubiquitous protein element consisting of two or more amphiphilic α-helices that have been coiled to form a supercoiled bundle (Lupas and Gruber, 2005). The main characteristic of the amphiphilic α-helix coiled-coil is the repeating heptad motif abcdefg, where positions a and d are mainly occupied by hydrophobic-hydrophilic residues, and positions b, c, e, f, and g are mainly occupied by hydrophilic residues. Each α-helix contains 3.6 residues per coil, which means that the repeating heptad motif places the residues at positions a and d on the same plane of the helix structure. This facilitates the formation of a highly stable supercoil, where the hydrophobic residues face inward towards each other within what is called a hydrophobic core, while the hydrophilic residues face outward. While the hydrophobic core of a coiled-coil protein typically contains mainly hydrophobic residues, not all residues in the core structure need to be hydrophobic. Other residues located within the core structure, such as polar residues, may be present, yet coiled-coil proteins that can still maintain their coiled-coil structure are known.

[0036] The oligomeric proteins described herein are based on variants of the leucine zipper sequence of the protein GCN4, known as GCN4-pIL. Here, GCN4-p'ad' refers to the amino acids located at positions a and d in the heptad motif. It has been demonstrated that by mutating the hydrophobic core residues at positions a and d, specifically by changing the ratio of leucine to isoleucine residues at these positions, it is possible to change the preferred oligomeric state of the protein structure from a dimer to a trimer (GCN4-pII) and a tetramer (GCN4-pLI) (Harbury et al., 1993; Delano and Bruenger, 1994).

[0037] The stability of these coiled-coil elements and their tendency to form oligomers led to the use of GCN4 coiled-coil structures as chimeric extensions (i.e., fusion partners) that induce oligomerization and stabilize the oligomeric structure in fusion proteins while simultaneously increasing the solubility of such proteins. In this regard, the inventors initially intended to investigate the putative interaction between LPS and two domains belonging to the trimer autotransporter adhesin SadA. To investigate this protein, two SadA constructs, K9 and K14, were prepared (see Example 1 below), both of which were stabilized by an adjacent GCN4-pII segment. Surprisingly, however, the GCN4-pII adapter used to stabilize the SadA construct was K in the nanomolar concentration range relative to LPS. D It was found to exhibit an extremely high affinity, possessing [specific characteristic].

[0038] Following this serendipitous discovery, the inventors developed an oligomeric protein with a coiled-coil structure based on the GCN4-pII protein that can be used as a binder for LPS. Further experiments revealed that the interaction between this protein and LPS arises from the binding of this protein to the lipid A component of LPS. As described above, the structure of the lipid A component is highly conserved among Gram-negative bacterial species, and therefore this oligomeric protein is understood to be able to bind to a wide range of bacterial endotoxins with extremely high affinity. Furthermore, this oligomeric protein can be overexpressed by recombinant methods in typical expression systems and can be purified from inclusion bodies without interacting with any natural endotoxins, which enables large-scale, sustainable, and cost-effective production.

[0039] The oligomeric proteins described herein comprise at least two monomer peptides. These monomer peptides represent individual subunits that constitute the oligomeric protein as a whole. Each monomer can form an α-helix. These monomers may be provided as separate peptides in the sense of separate peptide chains or chains that interact to form an oligomeric protein together. Thus, in such embodiments, peptide monomers may be considered as individual subunits of a protein, i.e., separate monomer peptide units. Thus, in some embodiments, each α-helix in an oligomeric protein may be considered to correspond to a separate monomer.

[0040] In other embodiments, monomer peptides may be linked together. Thus, monomer peptides may be linked or bound by linker sequences. In such embodiments, the oligomeric protein has a single-chain form in terms of its primary structure or sequence, but of course the monomer peptides can be seen as having “chains” that interact to form a coiled-coil structure. In such embodiments, monomer peptides may be considered as domains of a single-chain protein sequence. More specifically, the oligomeric protein may be seen as having a 3D structure composed of monomer peptide domains.

[0041] Each monomeric peptide contains at least one core sequence having at least 60% sequence identity with the heptad repeat sequence of SEQ ID NO: 1. SEQ ID NO: 1 represents a variant of the sequence of the model peptide GCN4-pII, which contains a repeat heptad motif abcdefg, based on a sequence derived from a dimerization motif at the C-terminus of the GCN4 protein, with isoleucine residues at positions a and d in the motif as shown below. In some embodiments, the core sequence may have at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1. In some embodiments, the core sequence may contain or consist of the sequence of SEQ ID NO: 1.

[0042] Sequence identity may be determined by any suitable means known in the art, for example, by using FASTA pep-cmp with a variable pamfactor, a gap creation penalty of 12.0, a gap extension penalty of 4.0, a 2-amino acid window, and the SWISS-PROT protein sequence databank. Other programs for determining amino acid sequence identity include the BestFit program, version 10 software package from the Genetics Computer Group (GCG) of the University of Wisconsin. This program uses the Smith-Waterman local homology algorithm, with default values ​​of a gap creation penalty of -8, a gap extension penalty of 2, an average match of 2.912, and an average mismatch of -2.003. In one embodiment, the comparison is performed over the entire length of the core sequence.

[0043] [Table 1]

[0044] Sequence ID 1 can be seen to contain several repeating abcdefg heptad motifs. As shown above, the a and d residues are I. However, as will be discussed in more detail below, the a and d residues can vary, and in one embodiment, the a and d residues may be I or L, or derivatives thereof, or other hydrophobic residues. As stated above, not all a and d residues in the heptad motif need to be hydrophobic. It is sufficient to have enough hydrophobic residues to form an oligomeric coiled-coil structure. This depends on the sequence context and the other residues present in the monomer peptide sequence.

[0045] In embodiments, the core sequence of each monomer peptide includes at least three heptad motifs abcdefg or variants thereof. Although the heptad motif is conveniently written as abcdefg, there is no requirement that the heptad repeat sequence in the core sequence begin at position a. This motif repeats with position a following position g, and therefore the heptad motif may begin at any position, provided that it includes all seven positions abcdefg in a consecutive order. Thus, for example, the motif defgabc is a valid heptad motif.

[0046] In some embodiments, the core sequence may contain one or more variants of the heptad motif abcdefg, each variant containing one or fewer insertions or deletions to the heptad motif. These variants of the heptad motif abcdefg containing insertions or deletions are collectively referred to as “variant motifs.” In this context, the terms “insertion” and “deletion” refer to the addition of a single residue to the heptad motif and the deletion of a single residue from the heptad motif, respectively.

[0047] Insertions or deletions can occur at any position within a heptad motif, including at either end of the heptad motif. For example, considering the insertion of residue X into the motif abcdefg, the resulting motifs could be Xabcdefg, aXbcdefg, abXcdefg, etc. Importantly, the representation of the remaining positions within the motif remains unchanged. This is true for both insertions and deletions. Therefore, for example, if a residue at position b is deleted, the remaining motif will contain the sequence acdefg.

[0048] Insertions or deletions of several consecutive residues are not considered a single insertion or deletion. Therefore, the at least three heptad motifs or variant motifs present in each core sequence must not contain more insertions and deletions to heptad motifs abcdefg than the total number of variant motifs present. Insertions or deletions adjacent to each other in a core sequence are permissible only if they are at adjacent ends of consecutive variant motifs and each consecutive variant motif contains only one insertion or deletion. In such cases, adjacent insertions / deletions can be seen as the product of two separate insertions / deletions in two separate variant motifs.

[0049] In some embodiments, the core sequence includes at least four heptad motifs or variant motifs. In some embodiments, the core sequence includes three to five heptad motifs or variant motifs. For example, the core sequence may include three, four, or five heptad motifs or variant motifs. In some embodiments, the core sequence may include at least three heptad motifs and no variant motifs. In other embodiments, the core sequence may include at least three variant motifs and no heptad motifs. Furthermore, the core sequence may include any combination of at least three heptad motifs and variant motifs, and these heptad motifs and variant motifs may be arranged in any order.

[0050] As discussed above, the coiled-coil protein structure depends on the organized arrangement of hydrophobic residues within the repeating heptad motif present in each α-helix. The hydrophobic residues within each α-helix are positioned to be presented primarily on a single face of that α-helix. In other words, the residues within each α-helix are positioned such that the residues presented on one face of that α-helix are primarily hydrophobic. This allows the hydrophobic faces of each α-helix in the oligomeric protein to form a stable hydrophobic core at the center of the protein structure. While it is not essential that hydrophobic residues be present at both positions a and b within every repeat of the heptad motif, typically, the majority of these positions are occupied by hydrophobic residues. To facilitate this structure in coiled-coil oligomeric proteins as defined herein, in one embodiment, at least 50% of the amino acid residues corresponding to positions a and d of the heptad motif or its variants within the core sequence are hydrophobic residues. As shown in the above configuration, positions a and d of the heptad motif in SEQ ID NO: 1 are represented by positions 4, 8, 11, 15, 18, 22, 25, and 29 of the sequence. Therefore, by alternative definition, it can be seen that at least 50% of the amino acid residues at the positions corresponding to positions 4, 8, 11, 15, 18, 22, 25, and 29 in SEQ ID NO: 1 are hydrophobic residues. Thus, in one embodiment, at least four of the eight "a" or "d" positions in the heptad repeat sequence of SEQ ID NO: 1 are hydrophobic residues.

[0051] More specifically, at least 52.5%, 55%, 60%, 62.5%, or 70% of the amino acid residues corresponding to positions a and d of the heptad motif in the heptad repeat sequence of SEQ ID NO: 1 are hydrophobic. However, in one typical embodiment, at least 75% of the amino acid residues corresponding to positions a and d of the heptad motif in the heptad repeat sequence of SEQ ID NO: 1 are hydrophobic. Therefore, in one embodiment, at least six of the eight "a" or "d" positions in the heptad repeat sequence of SEQ ID NO: 1 are hydrophobic residues.

[0052] A typical example is that in the sequence of Sequence ID No. 1, at least four, five, six, or seven of the 'a' or 'd' residues, i.e., the residues corresponding to positions 4, 8, 11, 15, 18, 22, 25, and 29 in Sequence ID No. 1, may be hydrophobic residues.

[0053] Based on knowledge of coiled-coil protein structures and sequences, modifying the sequence to obtain a coiled-coil structure based on a modified or mutant peptide of Sequence ID No. 1, including substitutions of residues at positions a and d in the heptad motif, would be within the scope of the routine knowledge of those skilled in the art.

[0054] As used herein, the term “hydrophobic residue” includes any amino acid residue recognized or identified as hydrophobic in the art. Such amino acids include the following protein-constituting amino acids: leucine, isoleucine, valine, alanine, methionine, phenylalanine, proline, and glycine. However, in embodiments, the hydrophobic residue is selected from the amino acids leucine, isoleucine, valine, alanine, methionine, phenylalanine, or their chemical derivatives. In another embodiment, the hydrophobic residue is selected from leucine, isoleucine, valine, alanine, and methionine, as well as chemical derivatives of these amino acids. Hydrophobic residues present in the core sequence may also include unconventional hydrophobic amino acids, i.e., hydrophobic amino acids having side chains not encoded by the standard genetic code. More specifically, these include fluoro derivatives of these amino acids, such as fluoroisoleucine and fluoroleucine. Other known derivatives include seleno derivatives, such as selenomethionine. Further examples of such unconventional hydrophobic amino acids are listed in Table 1 below, including D-amino acid variants (where all amino acids may be D-amino acids if a D-amino acid is included), LN-methylamino acid variants, D-α-methylamino acid variants, and DN-methylamino acid variants of the conventional hydrophobic amino acids defined above.

[0055] [Table 2-1]

[0056] [Table 2-2]

[0057] In some embodiments, at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the amino acid residues corresponding to positions a and d of the heptad motif or its variants are hydrophobic residues. In other words, at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the amino acid residues at positions 4, 8, 11, 15, 18, 22, 25, and 29 of SEQ ID NO: 1 are hydrophobic residues.

[0058] In some embodiments, 100% of the amino acid residues corresponding to positions a and d of the heptad motif or its variants are hydrophobic residues. In other words, 100% of the amino acid residues at positions 4, 8, 11, 15, 18, 22, 25, and 29 of SEQ ID NO: 1 are hydrophobic residues.

[0059] The hydrophobic residues within the core sequence may all be the same or may be different from one another. In some embodiments, each hydrophobic residue in the heptad motif or mutant motif is independently selected from the group consisting of leucine, isoleucine, valine, alanine, methionine, and their chemical derivatives, including their fluoro derivatives. In one embodiment, each hydrophobic residue in the heptad motif or mutant motif is independently selected from the group consisting of leucine, isoleucine, valine, methionine, and their chemical derivatives, including their fluoro or seleno derivatives. In one embodiment, each hydrophobic residue in the heptad motif or mutant motif is independently selected from the group consisting of leucine, isoleucine, and their chemical derivatives, such as fluoroleucine and fluoroisoleucine.

[0060] In some embodiments, at least 50% of the hydrophobic residues in the heptad motif or mutant motif are isoleucine or fluoroisoleucine. In some embodiments, at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the hydrophobic residues in the heptad motif or mutant motif are isoleucine or fluoroisoleucine. In some embodiments, 100% of the hydrophobic residues in the heptad motif or mutant motif are isoleucine or fluoroisoleucine.

[0061] Residues that do not form part of the hydrophobic core of the coiled-coil structure, i.e., residues at positions b, c, e, f, and g, are generally closer to the protein surface and therefore more exposed to the environment. It is not important what these residues are, and these residues may vary. In some embodiments, at least 50% of the amino acid residues corresponding to positions b, c, e, f, and g are polar residues. In some embodiments, at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the amino acid residues corresponding to positions b, c, e, f, and g are polar residues. In some embodiments, 100% of the amino acid residues corresponding to positions b, c, e, f, and g are polar residues.

[0062] As used herein, the term “polar residue” includes any amino acid residue recognized or identified as polar in the art. This term includes charged amino acids. Polar amino acid residues may be selected from the amino acids serine, threonine, asparagine, glutamine, aspartic acid, glutamic acid, histidine, arginine, lysine, tyrosine, cysteine, tryptophan, methionine, and chemical derivatives of these amino acids. In one embodiment, polar amino acid residues may be selected from the amino acids serine, threonine, asparagine, glutamine, aspartic acid, glutamic acid, histidine, arginine, lysine, and tyrosine. Furthermore, polar residues present in the core sequence may also include unconventional polar amino acids, i.e., polar amino acids having side chains not encoded by a standard genetic code. Examples of such unconventional polar amino acids, including D-amino acid variants, amide-isoster variants (such as N-methylamide, retro-inverse amide, thioamide, thioester, phosphonate, ketomethylene, hydroxymethylene, fluorovinyl, (E)-vinyl, methyleneamino, methylenethio, or alkane), LN-methylamino acid variants, D-α-methylamino acid variants, and DN-methylamino acid variants of the conventional polar amino acids defined above, are listed in Table 2 below. As described above, when D-amino acids are used, all amino acids in the monomer peptide may be D-amino acids.

[0063] [Table 3-1]

[0064] [Table 3-2]

[0065] While the consistent arrangement of hydrophobic and polar amino acids within a heptad motif is responsible for the structure of coiled-coil proteins, the general rules regarding residue positions are not immutable. That is, not all residues corresponding to positions a or d within the core sequence's heptad motif or variant motif are hydrophobic, nor do all residues corresponding to positions b, c, e, f, or g within the core sequence's heptad motif or variant motif have to be polar. In some embodiments, at least 5% of the amino acid residues corresponding to positions b, c, e, f, and g may be aliphatic residues. In some embodiments, at least 10% or at least 15% of the amino acid residues corresponding to positions b, c, e, f, and g may be aliphatic residues.

[0066] As used herein, the term "aliphatic residue" includes the amino acids glycine, alanine, isoleucine, leucine, proline, valine, and methionine, as well as chemical derivatives of these amino acids, particularly fluoroleucine and fluoroisoleucine, and their fluoro derivatives. Furthermore, aliphatic residues present in the core sequence may also include unconventional aliphatic amino acids, i.e., aliphatic amino acids having side chains not encoded by the standard genetic code, such as D-amino acid variants and other unconventional aliphatic amino acids.

[0067] The core sequence may include a specific percentage of polar residues and a specific percentage of aliphatic residues as defined above. For example, in some embodiments, at least 50% (or more than 50% as defined above) of the amino acid residues corresponding to positions b, c, e, f, and g may be polar residues, and at least 5% (or more than 5% as defined above) of the amino acid residues corresponding to positions b, c, e, f, and g may be aliphatic polar residues. However, this is not essential and may vary as described above.

[0068] In some embodiments, the core sequence as defined herein may be in contact with flanking amino acid sequences on one or both sides. When the core sequence is sandwiched on both sides, the flanking sequence on one side of the core sequence may be the same as or different from the flanking sequence on the other side of the core sequence. The flanking sequence may or may not form part of the coiled-coil structure of the oligomeric protein. Thus, the flanking sequence may contribute to or be part of the α-helix structure of the monomer peptide, and / or contribute to or form part of the coiled-coil structure in other ways, or be another part of the monomer peptide sequence. The flanking sequence may be used to perform various functions or to confer characteristics to the oligomeric protein. For example, the flanking sequence may be used to extend the heptad repeat sequence of the monomer peptide, to assist in the oligomerization of the monomer peptide, to link monomer peptides (e.g., within a single-stranded construct), or to provide a distinct functional portion to the oligomeric protein.

[0069] The length of the flanking sequence is not critical and may vary depending on the need and desire, or nature and / or purpose of the flanking sequence. For example, the length of a flanking sequence may be 1 to 300 amino acids, e.g., any one of 2, 3, 4, 5, 6 or 7 amino acids, to any one of 270, 250, 240, 230, 220, 210 or 200 amino acids. These ranges are shown only as examples and there are no restrictions on the length of the flanking sequence. In some embodiments implemented, the flanking sequence may be up to 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 amino acids. In some embodiments, shorter flanking sequences of up to 20, 15, 12, 10, 8, 7 or 6 amino acids are preferred.

[0070] Therefore, in some embodiments, the flanking sequence may include one or more heptad motifs and / or a portion of one or more heptad motifs. In this situation, the portion of a heptad motif may include one, two, three, four, or five residues that constitute a contiguous portion of the heptad motif. In some embodiments, the heptad motif in the flanking sequence corresponds to a heptad motif found in SEQ ID NO: 1 or a sequence having at least 60% (e.g., at least 70%, 80%, 90%) sequence identity thereto, provided that at least one of the amino acid residues corresponding to positions a and d of the heptad motif is a hydrophobic residue. In some embodiments, the flanking sequence may include SEQ ID NO: 1 or a portion thereof, or a sequence having at least 60% (e.g., at least 70%, 80%, 90%) sequence identity thereto. Furthermore, in such embodiments, at least 50% (e.g., at least 75%) of the amino acid residues corresponding to positions a and d of the heptad motif or its variants in SEQ ID NO: 1 are hydrophobic residues.

[0071] When a flanking sequence contains one or more heptad motifs, or a portion of one or more heptad motifs, such portions may be viewed as a continuation of the heptad motifs in the core sequence. As a result, the α-helix of the monomer protein that forms part of the coiled-coil structure of the oligomeric protein may extend beyond the end of the core sequence.

[0072] In some embodiments, the heptad motifs of the core sequence and the flanking sequence are contiguous. That is, the first residue of the flanking sequence (i.e., the residue directly adjacent to the end of the core sequence) corresponds to the position of the next heptad motif corresponding to the adjacent terminal residue of the core sequence. In this way, the repeating heptad motif abcdefg is maintained, and there is no gap between the heptad motifs of the core sequence and the flanking sequence.

[0073] In other embodiments, the flanking sequence may contain one or more heptad motifs that are not entirely contiguous with the heptad motif of the core sequence. That is, there may be one or more residues between the heptad repeats in the core sequence and the heptad repeats in the flanking sequence that do not form part of a contiguous repeating heptad motif.

[0074] In some embodiments, the core sequence and the flanking sequence may be arranged such that each monomer peptide does not contain more than 8 repeats of the heptad motif. In some embodiments, the monomer peptide does not contain more than 7, more than 6, or more than 5 repeats of the heptad motif. In other words, the monomer peptides of an oligomeric protein may contain up to 8, 7, 6, or 5 heptad repeats.

[0075] In some embodiments, the flanking sequence of the monomer peptide may not fully form a continuous α-helix with the core sequence and therefore may not be fully part of the coiled-coil structure of the oligomeric peptide.

[0076] In some embodiments, the oligomeric protein as defined herein may be in the form of a conjugate or fusion with one or more additional components or parts. As will be shown in more detail below, the oligomeric protein may be in the form of a conjugate with a detection part, an oligomerization part, or an immobilization part, or actually any desired component or part, such as a functional or structural component or part. The conjugated part may have any chemical or physical properties, such as small molecule or macromolecule. The oligomeric protein may be in the form of a fusion protein with a fusion partner. Thus, the detection part, the immobilization part, or other additional part may be, in effect, proteinaceous. That is, such a part may not even be a polypeptide component (in this specification, the term “polypeptide” is used to include any peptide, polypeptide, or protein, regardless of length). The oligomerization part may be a polypeptide. Furthermore, the oligomeric protein may be immobilized on a solid substrate. Thus, in some embodiments, one or more additional components may be a detection part, an oligomerization part, an immobilization part, or a fusion partner.

[0077] In some embodiments, one or more additional components of the oligomeric protein with which it is conjugated or fused may form all or part of a flanking sequence in one or more monomer peptides that make up the oligomeric protein. In other embodiments, the conjugated portion may be a separate component (i.e., separate from the oligomeric protein or its monomer peptides).

[0078] It will be understood that the presence of additional components within a flanking sequence may be in addition to, or substitute for, the presence of one or more heptad motifs within the same flanking sequence. That is, a given flanking sequence may contain both one or more heptad motifs or parts thereof and one or more additional components. If a flanking sequence actually contains one or more heptad motifs or parts thereof and one or more additional components, the flanking sequence may be arranged such that one or more heptad motifs or parts thereof are closer to the core sequence than the one or more additional components.

[0079] In some embodiments, the oligomeric protein is in the form of a fusion or conjugate with a single additional component. The additional component may form all or part of the flanking sequence in one of the monomer peptides. Alternatively, the oligomeric protein may be in the form of a fusion or conjugate with two or more additional components. In some embodiments, the additional components may form all or part of the same flanking sequence in the same monomer peptide. In some embodiments, a single monomer peptide may include a core sequence sandwiched on both sides by flanking sequences, each flanking sequence containing one or more additional components. Furthermore, the oligomeric protein as defined herein may include several monomer peptides, each containing one or more additional components in any of the arrangements shown above.

[0080] In the case of oligomeric proteins in the form of a conjugate with an oligomerization moiety, the oligomerization moiety may consist of several oligomerization sequences, and each monomer peptide contains an oligomerization sequence. Therefore, an oligomeric protein may consist of at least two monomer peptides, and each monomer peptide contains an oligomerization sequence in its flanking sequence.

[0081] The coiled-coil structures of oligomeric proteins disclosed herein may spontaneously form when monomer peptides are brought into contact with each other. Alternatively, the formation of oligomeric structures may require a "trigger" to overcome kinetic barriers and bring the monomer peptides together. Furthermore, in some embodiments, it may be necessary to stabilize the oligomeric coiled-coil structure of a protein. This initiation and stabilization of the oligomeric coiled-coil structure may be achieved by an oligomerization sequence. An oligomerization sequence is a protein sequence that can interact with other copies of itself in order to oligomerize, that is, to form an oligomer. It will be understood that if oligomerization is cooperative, that is, if a particular part of a larger protein can oligomerize easily and stably, this may help induce oligomerization in the rest of the protein structure that would not otherwise occur. For example, it is known in the art that the head domain of an adherent protein, e.g., the YadA head domain, can induce the formation of coiled-coil structures that other domains are not stable enough to form. The GCN4 protein was also used in a similar manner to stabilize the trimer autotransporter attachment factor (Hartmann et al., 2012). Therefore, this domain or other equivalent domains known in this field may be used as oligomeric sequences. As described above, when an oligomeric protein is conjugated with an oligomerization moiety, each monomer peptide within the oligomeric protein may contain an oligomerization sequence.

[0082] Furthermore, or in some embodiments, the initiation and stabilization of the coiled-coil structure may be performed by linking monomer peptides together.

[0083] While monomer peptides as defined herein can be considered to some extent in isolation, in some embodiments, two or more monomer peptides within the oligomeric proteins disclosed herein may be linked together. Thus, a flanking sequence may include one or more linker sequences. A flanking sequence may be added to or replaced by one or more heptad motifs or parts thereof, and one or more additional components that may be included in the flanking sequence. It will be understood that a flanking sequence may include any combination of heptad motifs and / or parts thereof, one or more additional components, and / or one or more linker sequences.

[0084] A linker sequence can link one monomer peptide to another so as to form a single peptide chain within at least a portion of an oligomeric protein. When two monomer peptides are linked via a linker sequence, it can be thought that one monomer peptide (i.e., the first monomer peptide) contains a flanking sequence that includes the entire linker sequence, and the linker sequence is directly attached to the core sequence of the other monomer peptide (i.e., the second monomer peptide does not have a flanking sequence at its end of the core sequence). Alternatively, the linkage between the two monomer peptides can be thought of as consisting partly of the flanking sequence of the first monomer peptide and partly of the flanking sequence of the second monomer peptide (i.e., both monomer peptides contain a flanking sequence that includes the linker sequence).

[0085] When a monomer peptide contains a linker sequence to link the monomer peptides together, it is sometimes advantageous that the flanking sequence between the two monomer peptides contains nothing other than the linker sequence and, optionally, a heptad repeat motif or a part thereof. However, the flanking sequence at either end of the linked monomer peptide chain may contain additional sequences (e.g., those considered above). In other words, in such linked, e.g., single-chain constructs, the oligomeric protein may be in the form of a conjugate with the additional part. To put it another way, the additional part may not be part of the monomer peptide, but rather conjugated to it.

[0086] Linker sequences can be of variable length and / or sequence. It can be understood that the linker sequence must be long enough to allow the helices formed by monomer peptides to assemble into coiled coils. However, there are no functional constraints on the maximum length of the linker sequence. Therefore, the linker sequence can be at least two residues long, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, or 30 residues long.

[0087] For example, in other embodiments, the linker sequence may contain 2 to 60 residues, particularly 5 to 55, 10 to 50, 15 to 45, or 20 to 40 residues. In one embodiment, the linker sequence may contain 2 to 50, 3 to 40, 4 to 30, 5 to 20, or 6 to 15 residues. The properties of the residues present in the linker sequence are not important. The residues may be any amino acid, such as a neutral amino acid or an aliphatic amino acid, or a polar amino acid, or a charged amino acid, or a structure-forming amino acid, such as proline. In embodiments, the linker sequence is a flexible linker sequence. Various flexible linkers that may be used are known and widely described in the art. As a typical example, at least 70% of the amino acids in the linker sequence may be selected from glycine, serine, threonine, alanine, proline, histidine, asparagine, aspartic acid, glutamine, glutamic acid, lysine, arginine, or derivatives thereof. In some embodiments, the linker is a glycine-rich sequence or a glycine-serine-rich sequence.

[0088] Each monomer peptide contains at least one core sequence, as defined above. In some embodiments, one or more monomer peptides may contain two or more core sequences, which may be the same or different from one another. As shown above, each core sequence may be in contact with a flanking sequence on one or both sides. If a core sequence is flanked on both sides, the two flanking sequences may be the same or different. Thus, each monomer peptide may contain two or more core sequences, and each core sequence may be flanked by a flanking sequence on one or both sides. Therefore, if a monomer peptide contains two or more core sequences, it will be understood that the monomer peptide may further contain up to two flanking sequences per core sequence. Thus, a monomer peptide containing two core sequences may contain up to four flanking sequences. For example, a monomer peptide may be arranged as FCFFCF, where F represents a flanking sequence and C represents a core sequence.

[0089] In some embodiments, the monomer peptide may contain two core sequences and three flanking sequences in an FCF configuration. In other embodiments, the monomer peptide may contain two core sequences and two flanking sequences in an FCCF configuration. In other embodiments, the monomer peptide may contain two core sequences and a single flanking sequence in a CCF configuration. In some embodiments, the monomer peptide may contain a single core sequence sandwiched on both sides by flanking sequences in an FCF configuration, or a single core sequence and a single flanking sequence. In some embodiments, the oligomeric protein may contain only one monomer peptide containing a flanking sequence. In some embodiments, each monomer peptide in the oligomeric protein consists of only one core sequence.

[0090] Each core sequence as defined herein has at least 60% identity with Sequence ID No. 1, which contains 30 residues, and it will be understood that each core sequence may have a length of 18 to 42 residues. In some embodiments, the core sequence in the monomer peptide may have a length of 19 to 41 residues, e.g., 20 to 40, 21 to 39, 22 to 38, 23 to 37, 24 to 36, 25 to 35, 26 to 34, 27 to 33, 28 to 32, or 29 to 31 residues. In some embodiments, the core sequence may contain 30 residues. There may be two or more core sequences. Each core sequence may be in contact with a flanking sequence on one or both sides. As described above, the flanking sequence may contain one or more heptad motifs or parts thereof, one or more additional components, and / or one or more linker sequences. Thus, in some embodiments, the monomer peptide as a whole may be significantly longer than the core sequence. In some embodiments, the monomer peptide may contain 24 to 1000 residues, for example, 24 to 900, 24 to 800, 24 to 700, 24 to 600, 24 to 600, 24 to 500, 24 to 400, 24 to 300, 24 to 250, 24 to 200, 24 to 150, 24 to 100, 24 to 75, 24 to 50, or 24 to 40 residues.

[0091] The oligomeric proteins as defined herein have a coiled-coil structure comprising at least two monomer peptides. As described above, this oligomeric protein is based on the C-terminal segment of the GCN4 transcription factor. The wild-type C-terminal GCN4 sequence forms a dimeric coiled-coil structure, i.e., a structure comprising two monomer peptides. However, it has been observed that by altering the residues at positions a and d within the heptad motif in the individual monomer peptides, the oligomeric state of the protein as a whole can be altered to form a trimer or tetramer structure. In some embodiments, the oligomeric protein is dimeric, trimer, or tetramer; that is, the oligomeric protein comprises two, three, or four monomer peptides. In preferred embodiments, the oligomeric protein is trimer.

[0092] Each monomer peptide within an oligomeric protein may be the same or different. This includes not only the sequence of the core sequence but also the presence or absence of one or more flanking sequences. In some embodiments, an oligomeric protein may contain two or more monomer peptides having the same core sequence. In some embodiments, an oligomeric protein may contain two or more completely identical monomer peptides. In some embodiments, all monomer peptides in an oligomeric protein may be identical. While the monomer peptides within an oligomeric protein do not need to be identical to one another, it is preferable that there be only minimal variation among the monomer peptides.

[0093] In some embodiments, each monomer peptide within an oligomeric protein may be provided as a separate peptide chain. In this case, each monomer peptide may be considered a physically separate subunit of the oligomeric protein complex. Alternatively, in some embodiments, two or more monomer peptides may be linked together. As outlined above, individual monomer peptides may be linked together by one or more linker sequences to form a single peptide chain, i.e., one end of a first monomer peptide may be linked to one end of a second monomer peptide. In some embodiments, all monomer peptides may be linked together to form a single peptide chain. In this case, the monomer peptides may be considered as individual domains of a single-chain, multi-domain protein construct.

[0094] Furthermore, monomer peptides may be linked together by chemical crosslinking in the form of one or more chemical crosslinks between them. Several methods for linking individual peptides by forming covalent bonds are known in the art, and any suitable such chemical crosslinking method may be used to link two or more monomer peptides in an oligomeric protein. For example, two or more monomer peptides may be linked by one or more disulfide bonds between specific cysteine ​​residues in the monomer peptides. Alternatively, two or more monomer peptides may be linked probabilistically by using a crosslinking agent such as formaldehyde, which can promote the formation of covalent bonds with lysine residues present in the monomer peptides. In some embodiments, the oligomeric protein may include a combination of monomer peptides linked together in the form of a single peptide chain and / or by chemical crosslinking, and several unlinked monomers provided on separate peptide chains.

[0095] The oligomeric proteins disclosed herein may be produced synthetically, for example, by linking amino acids or synthetically produced small peptides, or by recombinant expression of nucleic acid molecules encoding the protein or one or more monomer peptides. Such nucleic acid molecules may be produced synthetically by any suitable means known in the art. Therefore, the oligomeric proteins may be recombinant, synthetic, or artificial oligomeric proteins.

[0096] The oligomeric proteins as defined herein are provided as binders for binding to LPS. As described above, lipopolysaccharides are essential components in the outer membrane of all Gram-negative bacteria. However, not all Gram-negative bacteria have exactly the same lipopolysaccharides in their outer membranes. As used herein, the terms "LPS" or "endotoxin" (used without distinction from "LPS" as described above) refer to any lipopolysaccharide present in the outer membrane of Gram-negative bacteria.

[0097] An advantage is that the oligomeric proteins, as defined herein, can bind to LPS with extremely high affinity. This high affinity allows the oligomeric proteins to effectively bind to LPS even when LPS is present at very low concentrations, and as a result, LPS can be detected and / or removed. In some embodiments, the oligomeric proteins are bound at nanomolar or picomolar concentrations, or even lower ranges of K D It binds to LPS. For example, in some embodiments, the oligomeric protein has a K content of 10 nM or less, e.g., 5 nM or less, 1000 pM or less, 750 pM or less, 500 pM or less, 250 pM or less, 100 pM or less, 50 pM or less, 10 pM or less, 5 pM or less, 1 pM or less, or 500 fM or less. D It binds to LPS. Therefore, the oligomeric peptides as defined herein may be able to detect LPS in a sample in which LPS is present at a concentration of at least 100 pM. In some embodiments, the oligomeric proteins can detect LPS in a sample in which LPS is present at a concentration of at least 75 pM, more specifically at at least 50 pM, at least 25 pM, at least 10 pM, at least 5 pM, at least 3 pM, at least 1 pM, at least 750 fM, at least 500 fM, at least 250 fM, or at least 100 fM.

[0098] While we do not wish to be bound by theory, the inventors believe that the binding of oligomeric proteins as defined herein to LPS depends on both the overall coiled-coil structure of the protein and the interaction between LPS and individual residues within the protein. In this regard, the presence of positively charged residues within the oligomeric protein may help increase the affinity for binding. Again, while we do not wish to be bound by theory, it is hypothesized that positively charged residues may be involved in electrostatic interactions with negatively charged phosphate groups in the lipid A region of LPS. Thus, in some embodiments, the oligomeric protein contains a total of at least six cationic residues within the core sequence of the monomer peptide. In some embodiments, the oligomeric protein may contain a total of at least seven cationic residues within the core sequence of the monomer peptide, e.g., at least eight, at least nine, at least ten, at least twelve, or at least fifteen cationic residues.

[0099] As used herein, the term “cationic residue” includes lysine, arginine, histidine, and any genetically unencoded or modified amino acid residues that are positively charged at pH 7.0. Suitable genetically unencoded or modified cationic residues include analogs of lysine, arginine, and histidine, such as homolysine, ornithine, diaminobutyric acid, diaminopimelic acid, diaminopropionic acid, homoarginine, trimethyllysine, trimethylornithine, 4-aminopiperidine-4-carboxylic acid, 4-amino-1-carbamimidylpiperidine-4-carboxylic acid, and 4-guanidinophenylalanine.

[0100] The aforementioned cationic residues may be present within at least one core sequence of a single monomer peptide, or they may be dispersed across the core sequences of several monomer peptides in an oligomeric protein. In some embodiments, each monomer peptide contains at least two cationic residues in its core sequence. In some embodiments, each monomer peptide contains at least three, at least four, or at least five cationic residues in its core sequence.

[0101] As discussed above, oligomeric proteins interact with LPS via the lipid A component. Accordingly, the use of oligomeric proteins as defined herein as binders for binding to lipid A is also provided herein. The term “lipid A” as used herein refers to the lipid A component of LPS, which comprises two phosphoglucosamine sugar molecules linked by β-1,6-links and having four oxygen-linked acyl chains and two nitrogen-linked acyl chains, and which can interact with the outer membrane of Gram-negative bacteria.

[0102] In some embodiments, as described above, the oligomeric protein as defined herein may be in the form of a conjugate or fusion with one or more additional components or parts. In particular, the oligomeric protein may be conjugated with a detection part or an immobilization part. The additional part may be in the form of a polypeptide, and therefore the oligomeric protein may be in the form of a fusion protein with a fusion partner. The fusion partner is a polypeptide component of the fusion protein, separate from the oligomeric protein. In some embodiments, the oligomeric protein may be immobilized on a solid substrate.

[0103] Oligomer proteins may be conjugated to any suitable detection moiety, i.e., any moiety capable of providing a detectable signal. The detection moiety may be considered a label and may be detectable directly or indirectly. In some embodiments, oligomer proteins may be conjugated to a directly detectable detection moiety. A directly detectable moiety is one that can be detected directly without the use of additional reagents. For example, suitable directly detectable detection moieties may include fluorescent molecules (e.g., fluorescent proteins or organic fluorescent substances), color-emitting moieties (e.g., colored molecules or nanoparticles), particles, such as gold or silver particles, quantum dots, radioisotope labels, chemiluminescent molecules, etc. In particular, any spectrophotometrically or spectroscopically detectable label may be used for directly detectable moieties. Detectable labels may be distinguishable by color, but any other parameters, such as size, charge, etc., may be used.

[0104] An indirectly detectable portion is one that can be detected by using one or more additional reagents, for example, a part of a signal-generating system composed of two or more components. For example, the detection portion may contain an enzyme such as horseradish peroxidase (HRP) that can catalyze a reaction that produces a detectable signal, such as a color change. Therefore, when the detection portion is brought into contact with a substrate for the enzyme, the reaction proceeds and a detectable signal is generated.

[0105] Oligomer proteins can be in the form of a fusion protein with a fusion partner. In some embodiments, the fusion partner can be a detectable moiety. That is, an oligomer protein in the form of a conjugate with a detectable moiety may be considered equivalent to an oligomer protein in the form of a fusion protein with a detectable fusion partner. However, oligomer proteins can also be in the form of a fusion protein with a fusion partner other than a detectable moiety. In principle, the fusion partner can be any polypeptide, provided that the oligomer protein can still function as a binder for binding to LPS.

[0106] In some embodiments, oligomeric proteins may be immobilized on a solid substrate (i.e., a solid phase support or solid carrier). This immobilization may be achieved by any convenient method. Thus, the method or means of immobilization and the solid substrate may be selected, according to preference, from any number of immobilization means and solid substrates widely known in the art and described in the literature. In some embodiments, oligomeric proteins may be conjugated with an immobilization moiety to facilitate immobilization. The immobilization moiety may be directly conjugated to the solid substrate (e.g., chemically crosslinked). For example, in some embodiments, the immobilization moiety may contain cysteine ​​residues that can be coupled to cysteine ​​residues on the substrate in the form of disulfide crosslinks. In some embodiments, the immobilization moiety may be more indirectly conjugated to the substrate by a linker group or by one or more mediating groups. In some embodiments, the immobilization moiety may be, for example, its binding partner, i.e., a homozygous binding partner provided on the solid substrate, such as streptavidin or an antibody, such as biotin or a hapten. Therefore, oligomeric proteins may be covalently or acovalently linked to a solid substrate via an immobilization moiety. These links may be reversible (e.g., cleavable) or irreversible. In some embodiments, the links may be cleaved enzymatically, chemically, or photocatalyzed; for example, the links may be photosensitive.

[0107] In some embodiments, the interaction between the oligomeric protein and the solid substrate must be strong enough to allow the washing step to proceed; that is, the interaction between the oligomeric protein and the solid substrate must not be disturbed (or only slightly disturbed) by the washing step. For example, in one embodiment, less than 5% of the oligomeric protein is removed or eluted from the solid substrate in each washing step. In another embodiment, less than 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the oligomeric protein is removed or eluted from the solid substrate in each washing step.

[0108] The solid substrate may be any of the well-known substrates or matrices currently widely used or proposed for immobilization, separation, etc. These may take the form of particles (e.g., beads that may be magnetic, paramagnetic, or nonmagnetic), sheets, gels, filters, membranes, fibers, capillaries, slides, arrays, tips or microtiter strips, tubes, plates or wells, etc.

[0109] In some embodiments, the oligomeric protein is immobilized on beads or resin, or in or on wells or containers or columns or filter materials, or on the surface of a detection device.

[0110] The substrate may be made of glass, silica, latex, apatite, or polymer material. In some situations, materials with a high surface area may be particularly suitable. Such substrates may have irregular surfaces, such as porous or granular, e.g., particles, fibers, webs, sinter, or sieves. Granular materials, such as beads, especially polymer beads, are useful due to their larger binding capacity. It will be understood that these beads may be provided in any suitable configuration, as is well known in the art. For example, beads may be packed into columns, such as filtration columns.

[0111] Conveniently, the granular solid substrates used in this disclosure may include spherical beads. The size of the beads is not important, but they may have a diameter of, for example, on the order of at least 1 μm. In one embodiment, the beads may have a diameter of at least 2 μm. In one embodiment, the beads may have a maximum diameter not greater than 10 μm, for example, not greater than 6 μm. Monodisperse particles, i.e., those with substantially uniform size (e.g., with a diameter standard deviation of less than 5%), have the advantage of providing very uniform reaction reproducibility. Typical monodisperse polymer particles may be produced by the technique described in U.S. Patent Application Publication No. 4336173.

[0112] In some embodiments, the solid substrate may be a resin, such as an amylose resin. The resin may be supplied in any suitable form, such as a spin column filter or a flow column. In some embodiments, the oligomeric protein may be immobilized in or on a well or container, such as a multiwell plate.

[0113] In some embodiments, oligomeric proteins may be immobilized on the surface of a detection device, such as a chip or microarray. In this regard, the oligomeric proteins may bind to LPS and form a capture array or biosensor capable of detecting LPS. In some embodiments, the oligomeric proteins may be immobilized on a surface plasmon resonance (SPR) chip. Biosensors capable of measuring signals corresponding to the binding of a target to an immobilized capture protein are well known in the art, and the oligomeric proteins as defined herein may be provided in any suitable such device.

[0114] Therefore, it can be seen that the use of oligomeric proteins as defined herein as binders for binding to LPS may include the use of oligomeric proteins for detecting and / or removing LPS in or from a sample.

[0115] Therefore, the use of oligomeric proteins as defined and described herein includes, in particular, in vitro uses, i.e., uses in which LPS is bound, detected, or removed in vitro.

[0116] In this regard, the Specified Publicly Provided Methods for binding to LPS include contacting LPS or a sample containing LPS with an oligomeric protein as defined herein, thereby enabling the protein to bind to LPS in such a way that it forms a protein-lipopolysaccharide complex. The foregoing disclosures relating to the oligomeric protein used in binding to LPS will be understood to apply equally to methods for binding to LPS involving the same oligomeric protein.

[0117] In one embodiment, this method is an in vitro method.

[0118] As used herein, the term “sample” includes any sample that may contain or be contaminated with LPS, or is desired to be tested. Such samples include clinical samples derived from patients or more generally from subjects, environmental samples, and samples of products to be tested for endotoxin contamination. Clinical samples derived from patients may include any samples of bodily fluids or tissues, such as blood samples, lymph samples, saliva samples, urine samples, fecal samples, cerebrospinal fluid samples, or any other suitable biological samples taken from a patient. In a preferred embodiment, the clinical sample is a blood sample.

[0119] Samples of products to be tested for endotoxin contamination may originate from any product suspected of being contaminated with endotoxins, particularly those intended for human consumption or interaction. Such products include, for example, products from the pharmaceutical and medical industries, such as reagents, medical devices, equipment, consumables, drugs, and vaccines. Similarly, samples may originate from products in the food and beverage industries or from environmental samples, such as drinking water and groundwater.

[0120] In one embodiment, the sample may be a liquid sample containing a portion of the product to be tested, or it may be a sample derived from the surface of a product, such as a solid product, e.g., a medical device, or a surface, e.g., a surface in an operating room or another sterile environment, where endotoxin contamination is desired. Such a sample may include, for example, a swab or cleaning solution taken from the surface of the product.

[0121] In some embodiments, the method of binding to LPS is a method for detecting the presence of LPS in a sample, and this method is (a) The step of contacting the sample with an oligomeric protein as defined herein to enable the protein to bind to LPS so as to form a protein-lipopolysaccharide complex; (b) A step to detect the presence of a protein-lipopolysaccharide complex, Includes.

[0122] In some embodiments, the method of binding to LPS may be a method for detecting the presence of Gram-negative bacteria in a sample suspected of containing Gram-negative bacteria, and this method is (a) The step of contacting the sample with an oligomeric protein as defined herein to enable the protein to bind to LPS in the outer membrane of a Gram-negative bacterium so as to form a protein-lipopolysaccharide complex; (b) A step to detect the presence of a protein-lipopolysaccharide complex, Includes.

[0123] The step of detecting protein-lipopolysaccharide complexes may be carried out by any appropriate means known in the art. Protein-lipopolysaccharide complexes may be detected directly or indirectly. The appropriate method for detecting protein-lipopolysaccharide complexes may be selected depending on how the sample is brought into contact with the oligomeric protein.

[0124] The step of contacting the sample with the oligomeric protein may include applying the sample to a substrate on which the oligomeric protein is immobilized, as outlined above, and the substrate is configured to allow measurement of the binding of the sample to the oligomeric protein. In some embodiments, for example, the oligomeric protein may be immobilized on the surface of a detection device, such as an SPR chip or another suitable biosensor, which can detect the interaction between the sample and the oligomeric protein as outlined above. Thus, the step of contacting the sample with the oligomeric protein may include applying the sample to a solid substrate on which the oligomeric protein is immobilized.

[0125] In other embodiments, for example, oligomeric proteins may be immobilized in the wells of a multiwell plate to form an LPS assay. Such assays are well known in the art. When a sample is applied to a plate containing the immobilized oligomeric proteins, any LPS present in the sample is bound by the oligomeric proteins, and other components in the sample can be washed away. Thus, the step of contacting the sample with the oligomeric proteins includes applying the sample to a multiwell plate on which the oligomeric proteins are immobilized. Furthermore, a method for detecting the presence of LPS in a sample may further include a step of washing the protein-lipopolysaccharide complex before the detection step to remove unbound components of the sample and thus improve the accuracy of the method. Suitable reagents and protocols for such a washing step are well known in the art. The protein-lipopolysaccharide complexes retained in the plate can then be detected using any suitable detection moiety capable of binding to LPS. The detection moiety may be detectable directly or indirectly. As outlined in more detail below, the inventors have adapted an ELISA-like assay (ELITA) using the tail spike protein of salmonella phage, first reported by Schmidt et al. in 2016, to detect LPS. This assay uses a tail spike protein capable of binding to LPS and containing an N-terminal streptag, along with streptavidin-conjugated horseradish peroxidase, to detect protein-lipopolysaccharide complexes. A detectable color change is induced when the enzyme substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is added to the plate. Thus, the step of detecting the presence of protein-lipopolysaccharide complexes may involve contacting the protein-lipopolysaccharide complexes with a detection portion containing an enzyme capable of binding to LPS and catalyzing a reaction that generates a detectable signal, as well as a suitable substrate that induces such a detectable signal.

[0126] In some embodiments, the oligomeric protein may be in the form of a conjugate containing the detection moiety itself, as outlined above. Thus, the protein-lipopolysaccharide complex may be detected by detecting a signal from the detection moiety conjugated to the oligomeric protein. This detection may be performed by any method appropriate for detecting a signal from the detection moiety in question, for example, by using fluorescence microscopy to observe a fluorescent label conjugated to the oligomeric protein.

[0127] In some embodiments, the method of binding to LPS is a method of removing LPS from a sample, and this method is (a) The step of contacting the sample with an oligomeric protein as defined herein to enable the protein to bind to LPS so as to form a protein-lipopolysaccharide complex; (b) A step of separating the protein-lipopolysaccharide complex from the sample, Includes.

[0128] Here again, it will be understood that the step of separating the protein-lipopolysaccharide complex from the sample may be carried out by any suitable means known in the art, and that this means depends on how the sample is brought into contact with the oligomeric protein.

[0129] In some embodiments, the oligomeric protein may be immobilized on a solid substrate, and therefore the step of contacting the sample with the oligomeric protein may include the step of applying the sample to the solid substrate on which the oligomeric protein is immobilized. As described above, the oligomeric protein may be immobilized on any suitable substrate known in the art. Specifically, the solid substrate may be in the form of particles (e.g., beads), filters, or columns. Again, suitable reagents and protocols for using such substrates to separate the bound target molecule from the sample are well known in the art.

[0130] As described above, oligomeric proteins may be immobilized on beads, and these beads may be magnetic. The term "magnetic" as used herein means that a substrate may have a magnetic moment when placed in a magnetic field, and is therefore mobile under the influence of that magnetic field. In other words, a substrate containing magnetic particles may be readily removed by magnetic aggregation, which provides a rapid, simple, and efficient method for separating protein-lipopolysaccharide complexes from a sample once the complexes have formed.

[0131] In another embodiment, for example, the oligomeric protein may be immobilized on a resin packed into the column. In this example, when the sample is brought into contact with the oligomeric protein, i.e., when the sample is applied to the column, the LPS is bound by the oligomeric protein and retained in the column, while the rest of the sample passes through the column. In some embodiments, the method may include several steps of bringing the sample into contact with the oligomeric protein to ensure that all of the LPS is bound; that is, the sample may be applied to the column several times. Furthermore, the method may include a step of washing the protein-lipopolysaccharide complex before the separation step to avoid inadvertently removing other components from the sample in addition to the LPS; that is, the column may be washed with an appropriate reagent.

[0132] When binding to LPS, detecting the presence of LPS in a sample, or removing LPS from a sample, it is advantageous if the reagents involved in the binding, detection, or removal steps, particularly the oligomeric protein, can be reused. Therefore, the methods disclosed herein may further include the step of contacting the protein-lipopolysaccharide complex with at least one non-denaturing surfactant in order to remove LPS from the oligomeric protein, i.e., to cleave the protein-lipopolysaccharide complex so that the oligomeric protein can be reused.

[0133] In this regard, this specification provides a kit used as a binder for LPS as defined herein, or used in a method as defined herein, the kit is (i) with an oligomeric protein as defined herein; (ii) at least one non-denaturing surfactant, Includes.

[0134] Non-denatured surfactants are well known in the art, and those skilled in the art may use any suitable non-denatured surfactant. For example, at least one non-denatured surfactant may be selected from nonionic surfactants, anionic surfactants, cationic surfactants, or zwitterionic surfactants, or any combination thereof. In this regard, at least one non-denatured surfactant may have a head group selected from linear polyethylene glycol (PEG) group, polysorbate group, β-glycoside sugar group, N-methylglucamine group, N-oxide group, dimethylammonium-1-propanesulfonate group, carboxylic acid group, sulfate group, or quaternary amine group. At least one non-denatured surfactant may be selected from CHAPS, Zwittergent 3-12, Polysorbate 80, Polysorbate 20, Triton X-100, or any combination thereof. In some embodiments, at least one non-denatured surfactant may be a mixture of non-denatured surfactants. In some embodiments, the mixture of non-denatured surfactants comprises or consists of CHAPS, Twittergent 3-12, Polysorbate 80, Polysorbate 20, and Triton X-100.

[0135] It will be understood that the surfactant must be present at a concentration sufficient to cause the protein-lipopolysaccharide complex to split without reaching a concentration so high as to permanently weaken the function of the oligomeric protein. In some embodiments, the surfactant may be present at a total concentration of at least 0.1% (weight / weight) or 0.1% (volume / volume), i.e., the concentration of all surfactants present. In some embodiments, the concentration of the surfactant may be at least 0.15% (weight / weight) or at least 0.15% (volume / volume), for example, at least 0.2% (weight / weight) or at least 0.2% (volume / volume), at least 0.25% (weight / weight) or at least 0.25% (volume / volume), or at least 0.5% (weight / weight) or at least 0.5% (volume / volume). In one embodiment, at least one non-denatured surfactant includes a combination of 0.05% (weight / weight) CHAPS, 0.05% (weight / weight) Twittergent 3-12, 0.05% (volt / volt) Tween 80, 0.05% (volt / volt) Tween 20, and 0.05% (volt / volt) Triton X-100.

[0136] In further embodiments, products comprising oligomeric proteins immobilized on a solid substrate are provided herein, wherein the oligomeric proteins are as defined herein. The solid substrate may be any solid substrate disclosed herein. That is, the above disclosures relating to the use of oligomeric proteins immobilized on a solid substrate apply equally to the situation of products comprising oligomeric proteins immobilized on a solid substrate. In this regard, the solid substrate may be a sheet, gel, filter, membrane, fiber, capillary, slide, array, tip, microtiter strip, tube, plate, or well. In particular, the oligomeric proteins may be immobilized on the surface of a detection device, such as an SPR tip or biosensor.

[0137] It will be understood from the above disclosure that the oligomeric proteins described herein provide alternative binders for binding to LPS, which can address several problems associated with known methods for binding to and detecting LPS. More specifically, the oligomeric proteins described herein can degrade LPS aggregates. Therefore, these oligomeric proteins can reduce the effect of LPS masking caused by aggregation and thus effectively increase the measurable concentration of LPS in a sample. Accordingly, these oligomeric proteins support LPS detection methods that can detect low concentrations of LPS.

[0138] Furthermore, this detection method avoids challenges associated with the LAL assay, such as the expensive and unsustainable harvesting of amebosite lysates. Moreover, this method avoids all potential problems associated with the use of factor C, which can also exist with recombinant variants of the LAL assay. [Examples]

[0139] method Protein expression and purification Salmonella adhesin A (SadA) constructs sandwiched by a GCN4 adapter (shown in Figure 7) were produced as previously described (Alvarez et al., 2008; Hartmann et al., 2012). Transformed BL21-gold (DE3) cells were grown in 2 L of ZYP-5052 autoinduction medium (Studier, 2005), and overexpression was induced by adding 200 ng / mL of anhydrotetracycline (AHTC) at OD600=0.6 and then expressing the cells overnight at 30°C. Cells were pelleted at 6000 × g (Beckmann JLA8.1000 rotor) for 30 minutes and resuspended in 20 mL of Tris / HCl pH 7.4, 40 mM NaCl, and 5 mM MgCl2 containing 200 μL of EDTA-free protease inhibitor cocktail (Merck) and DNI. After resuspending, the cells were lysed by French press, and the resulting lysis solution was diluted in 50 mL of equilibrium buffer (20 mM Tris / HCl, pH 7.9, 5 M guanidine hydrochloride, 0.5 M NaCl, 10% glycerol). The solution was incubated at room temperature for 1 hour with stirring, and then centrifuged at 75,000 × g for 1 hour (Beckman Ti70 rotor) to remove all undissolved granular material. The resulting solution was placed on a 20 mL Ni Sepharose Excel column (GE Life Sciences) that had been pre-equilibriumized with equilibrium buffer. After applying the sample, the column was washed with four times the volume of equilibrium buffer, and eluted using a 0-100% gradient elution buffer (20 mM Tris / HCl, pH 7.5, 5 M guanidine hydrochloride, 0.5 M NaCl, 10% glycerol, 500 mM imidazole). The eluted fraction was analyzed by SDS-PAGE, and the fraction containing the target protein was pooled and refolded by two overnight dialysis cycles in 2 L of refolding buffer (20 mM MOPS, pH 7.4, 350 mM NaCl, 10% glycerol).

[0140] LPS production and purification LPS was induced by inoculating a 20 mL lysogenic broth (LB) preculture from a single bacterial colony (see Table 3 below for the strains used), and the cultures were grown overnight at 37°C.

[0141] [Table 4]

[0142] Six sets of 1L cultures in 2L baffled flasks were inoculated from the pre-culture and grown overnight at 37°C on a shaker. Bacteria were collected by centrifugation at 6000×g for 30 minutes (Beckmann JLA 8.1000 rotor). Subsequent purification was performed using two different methods depending on the type of LPS.

[0143] Rough LPS was purified using phenol-chloroform-petroleum ether extraction according to the protocol described by Galanos et al. (Galanos, Luederitz, and Westphal, 1969). After collection, the bacterial pellet was washed three times with 40 mL of ethanol and once with acetone, and then left overnight under an airflow. The dried pellet was homogenized using a mortar and pestle and dissolved in 40 mL of a mixture of 90% (g / vol) liquid phenol, chloroform, and petroleum ether in a 2:5:8 ratio. After incubation on a shaker for 1 hour, the undissolved material was pelletized at 4200 × g for 15 minutes, and the supernatant was collected. Chloroform and petroleum ether were removed under an airflow for 4 hours or until the phenol began to crystallize. The solution was resuspended by heating to 40°C, and water was added dropwise with stirring until the LPS precipitated (5 drops × 3 times). LPS was pelletized at 4200 × g for 15 minutes, and water was added to the supernatant to collect all remaining LPS. The pellets were washed twice with 10 mL of 80% (g / vol) phenol, then added to 20 mL of Milli-Q water and centrifuged at 100,000 × g for 1 hour (Beckmann, MLA-50 rotor). The final pellets were added to 50 mL of Milli-Q water and freeze-dried to obtain pure LPS.

[0144] Smooth LPS was purified according to the protocol described by Darveau et al. (Darveau and Hancock, 1983). The bacteria were washed twice and resuspended in 40 mL of 10 mM Tris-HCl pH 8.0 and 2 mM MgCl2. Dissolution was performed by French press, followed by further disruption by sonication. The resulting suspension was incubated overnight at 37°C with stirring with 200 μg / mL DNase I and 50 μg / mL RNase A. To 15 mL of the suspension, 5 mL of 10 mM Tris-HCl pH 8.0 containing 0.5 M EDTA, 2.5 mL of 10 mM Tris-HCl pH 8.0 containing 20% ​​SDS, and 2.5 mL of 10 mM Tris-HCl pH 8.0 were added, and LPS micelles were further disrupted by sonication. To pelletize the undissolved cellular components, the solution was centrifuged at 39,000 × g for 30 minutes at 20°C (Sorvall, SS-34 rotor), and the supernatant was lyophilized. The lyophilized crude extract was dissolved in a small amount of water, and LPS was precipitated overnight at -40°C using 2 volumes of ice-cold ethanol and 0.375 M MgCl2. The precipitated LPS was centrifuged at 11,000 × g for 15 minutes at 4°C (Sorvall, SLA3000 rotor), and the resulting pellet was resuspended in the same volume of 90% (g / vol) phenol with stirring at 65°C for 30 minutes. To accelerate phase separation, the mixture was centrifuged at 4,000 × g for 10 minutes. The aqueous phase was collected, and the phenol phase was extracted again with water. The separated aqueous phase was pooled, and phenol was extracted using 1 / 4 volume of chloroform. The aqueous phase was left under an airflow overnight to evaporate all remaining organic solvents, and then dialyzed against MQ water for 3 days using an MWCO500 dialysis membrane. To obtain pure LPS, the dialyzed LPS was freeze-dried.

[0145] The purity of the isolated LPS product was controlled by tricine-SDS-polyacrylamide gel electrophoresis (Marolda et al., 2006).

[0146] Preparation of antigen polysaccharides Polysaccharides were isolated from wild-type S. tifimulium (smooth) LPS by mild acid hydrolysis of the glycosidic bond linking lipid A to the proximal KDO sugar (Raetz and Whitfield, 2002a). 4-5 mg / mL of S. tifimulium LPS was dissolved in 10% acetic acid and incubated at 100°C for 1 hour. The resulting lipid A was removed from the solution by centrifugation at 10,000 × g for 30 minutes at 4°C, and the supernatant containing the polysaccharide was freeze-dried overnight.

[0147] ELISA-like tail spike adsorption (ELITA) assay The ELITA assay was first described by Schmidt et al. using whole bacteria (Schmidt et al., 2016). In this invention, the assay was modified to use purified protein in a Nunc MaxiSorp 96-well flatwell plate (shown in Figure 11). The wells were saturated by incubation overnight with 100 μl of either 10 μg / mL K9-His or K14-His in PBS buffer. After a 2-hour blocking step with 2% bovine serum albumin (BSA) in PBS, 100 μl of a dilution of Salmonella tiphimurium LPS ranging from 200 μg / mL to 0.0023 μg / mL was added as a binding partner and incubated for 1 hour. After adding 100 μL of P22 tail spike protein (P22TSP) with an N-terminal streptag (Strep-Tag) (registered trademark) II (IBA) for 1 hour, the wells were incubated with 100 μL of 1:10,000 streptactin-conjugated horseradish peroxidase (IBA, Göttingen) for 1 hour, developed with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, Sigma-Aldrich) for 30–60 minutes, and read at 407 nm using a plate reader. Between each of the above steps, the wells were washed three times with 150 μL of PBS buffer containing 0.1% BSA (Tween-20 was excluded in these experiments as it interferes with this assay). Error propagation was calculated by subtracting the average background signal (0 μg / mL LPS) from each average signal and adding the individual standard deviations for the triplets to the baseline in the Cartesian coordinate system.

[0148]

number

[0149] (Here, δQ is the uncertainty of the combination of sum Q). The data is processed using the following Hill formula.

[0150]

number

[0151] Here, Y is the percentage of occupied receptor binding sites, Ymax is the maximum binding, [L] is the concentration of the free ligand, and n is the number of binding sites. The dose-response curve and dissociation constant K are fitted to this curve. D The following calculations were performed. Each construct has two GCN4-PII motifs, but these motifs are located at opposite ends of this protein and are therefore not expected to cooperate, so n was treated as equal to 1. The average molecular weight of smooth S. tifimurum LPS was calculated to be 22 kDa, assuming an average of 30 O antigen repeat polysaccharide structures, as reported (Peterson and McGrowty, 1985; Laetz and Whitfield, 2002b; Schmidt et al., 2016).

[0152] Surface plasmon resonance experiments (Examples 1-3) All SPR experiments were performed at ambient temperature using a Reichert 2 SPR system with PBS-E (PBS pH 7.4 + 5 mM EDTA) running buffer. Proteins were diluted to 50 μg / mL in 20 mM sodium acetate buffer pH 4.5 and immobilized on a CMD200 sensor chip (Xantec Bioanalytics, Düsseldorf, Germany) using NHS-EDC amine coupling (Fischer, 2010) to produce a response of 2000–9000 μRIU. After comparisons of various reference compounds (ethanolamine, BSA, casein, and skim milk) (Peterfi et al., 2000), ethanolamine was selected as the standard coating for the reference channel for all experiments.

[0153] All ligands were dissolved in running buffer at a concentration of 1 mg / mL by extrusion (21 passes through a 100 μm filter at 70°C). Three sets of experiments were performed at a flow rate of 50 μL / min. Each sample was injected into both the measurement channel and the reference channel for 90 seconds, followed by dissociation for 300 seconds. The tip was regenerated by two 30-second injections of regeneration buffer (0.05% (wt / wt) CHAPS, 0.05% (wt / wt) Zwittergent 3-12, 0.05% (volt / volt) Tween 80, 0.05% (volt / volt) Tween 20, and 0.05% (volt / volt) Triton X-100) (Andersson, Areskoug, and Hardenborg, 1999). The measurement data was exported to TraceDrawer (RidgeView Instrument Lab) for processing, and the final curve was generated using Origin (OriginLab Corporation). The following equation

[0154]

number

[0155] Here, S is the normalized signal S0, and the signal for each construct was normalized to K9 using this signal.

[0156] Surface plasmon resonance experiments (Examples 4 and 5) SPR experiments were performed at ambient temperature on a Nicoya OpenSPR system using PBS-E (PBS pH 7.4 + 5 mM EDTA) running buffer. SadA K9 was diluted to 50 μg / mL in 10 mM sodium acetate buffer pH 4.5 and immobilized on a carboxyl sensor (OpenSPR) using NHS-EDC amine coupling (Fischer, 2010) to obtain a response of 700 RU.

[0157] All ligands were dissolved in running buffer at a concentration of 1 mg / mL by extrusion (21 passes through a 100 μm filter at 70°C). Three sets of experiments were performed at a flow rate of 35 μL / min. Each sample was injected into both the measurement channel and the reference channel for 125 seconds, followed by dissociation for 300 seconds. The tip was regenerated by injecting regeneration buffer (0.05% (wt / wt) CHAPS, 0.05% (wt / wt) Twittergent 3-12, 0.05% (volt / volt) Tween 80, 0.05% (volt / volt) Tween 20, and 0.05% (volt / volt) Triton X-100) (Anderssohn et al., 1999) for 125 seconds. Measurement data was exported to a trace drawer (RidgeView Instruments Lab) for processing, and the final graph was generated using Origin (Origin Lab Corporation).

[0158] Electron microscopy The sample was attached to a measurement grid, stained with 1% uranyl acetate for 1 minute, and embedded in 1.8% methylcellulose / 0.4% uranyl acetate. Images were recorded at 80kV using an Olympus Quemesa camera and a Philips CM100 transmission electron microscope.

[0159] Limulus amebosite lysate (LAL) assay The masking effect of GCN4-PII on LPS was tested using the LAL assay (Pierce, Thermofisher). GCN4-PII concentrations ranging from 200 μg / mL to 20 pg / mL were spiked with 0.5 endotoxin units (EU / mL) per mL of LPS, and developed according to the provided protocol.

[0160] Circular dichroism Spectra were recorded using a JUSCO J-810 spectropolar meter (JUSCO International Co., Ltd.). Measurements were performed using a 1.0 cm optical path feldspar cell. Each sample was scanned five times in the range of 190-250 nm using a bandwidth of 0.5 nm and a scan speed of 50 nm / min. Spectra were recorded at 37°C in 10 mM Tris, pH 7.4, with GCN4-pII ratios to LPS of 0, 0.5, 1, 3, and 9. The approximate α-helix content of the peptide was calculated using K2D2.

[0161] Nuclear magnetic resonance (NMR) spectroscopy NMR experiments for assignment were performed in a Bel-Art (trademark) SP Sciencware (trademark) 5mm outer diameter thin-walled precision NMR tube containing 1.5mM synthetic FMet-GCN4-PII (Genscript, China), 7% D2O, and 450 μL of 0.2mM 4,4-dimethyl-4-silapentanesulfonic acid (DSS). Spectra were acquired at 308K on a Bruker Avance II 600 MHz NMR spectrometer equipped with a 5mm 1H / 13C / 15N cryoprobe. DSS was used as the internal chemical shift standard, and 13C and 15N were referenced using the described frequency ratios (Wishart et al., 1995). The following spectra were collected for assignment purposes. 13C-1H-HSQC, 15N-1H-HSQC, 1H-1H COSY, 1H-1H TOCSY with mixing times of 60 and 80 ms, and 1H-1H NOESY with mixing times of 80 and 100 ms. All spectra were processed using Topspin 4.0, and peaks were selected using CARA 1.9.1 (Keller, 2004).

[0162] Biotin-LPS (B-LPS)-based ELISA Black 96-well Greiner microplates were coated with 100 μl of 10 μg / mL SadA K9 (Cold Spring Harbor) in PBS buffer by incubation at 4°C overnight. The following day, wells were blocked by incubation with 150 μl of 2% bovine serum albumin (BSA) in PBS. 100 μl of diluted biotinylated LPS in the range of 4 ng / mL to 0.06 ng / mL was added as a conjugate and incubated for 1 hour. The plates were washed three times with 150 μl of PBS + 0.1% BSA and 100 μl of 1:10,000 streptactin-conjugated horseradish peroxidase (IBA) for 1 hour, developed with QuantaRed fluorescent substrate (Thermo) for 15 minutes, and fluorescence was read at Ex: 550 nm and Em: 610 nm.

[0163] protocol Add 100 μL of 1.10 μg / mL SadA fragment solution and coat a 96-well black grainer / nummaxisoap plate by leaving it overnight at 4°C. 2. Empty the wells and block with 150 μL of 5% BSA in PBS for 2 hours. 3. Wash three times with 150 μL of 0.1% BSA in PBS. 4. Empty the well and add 100 μL of biotinylated LPS dilution. 5. Wash three times with 150 μl of 0.1% BSA in PBS. 6. Add 100 μL of streptactin-conjugated HRP (IBA) (1:20,000 dilution in PBS + 0.35 M NaCl, 50 mM MgSO4, and 0.1% BSA) for 60 minutes. 7. Wash four times with PBS + 0.35M NaCl, 50mM MgSO4, and 0.1% BSA (150μL), then wash once with PBS + 0.1% BSA. 8. Incubate with Quantared HRP-substrate and stop the reaction after 15 minutes. 9. Read the fluorescence at an excitation wavelength of 550 nm and an emission wavelength of 610 nm.

[0164] All substrates were prepared according to the seller's instructions. When subtracting the background from the signal, the propagation rate of error was calculated by adding the individual standard deviations for the repeats to the baseline of the orthogonal coordinates (δQ = √(δa 2 + δb 2 + ··· + δz 2 ), where δQ is the uncertainty of the combination of the sum Q). The error bars represent one standard deviation.

[0165] Example 1 - GCN4-PII binds to lipid A We intended to examine the putative interaction between LPS and two domains belonging to the trimeric autotransporter adhesin SadA. Two SadA constructs that have already been described (Alvarez et al., 2008; Hartmann et al., 2012), both of which are stabilized by flanking GCN4-PII segments, K9 and K14, were used. K9 or K14 was covalently linked to the SPR chip and various LPS components were injected. A schematic version of the LPS structure is provided in Figure 2 for reference.

[0166] The injection of smooth LPS immediately produced a response, and the response approached a steady state towards the end of the injection (Figure 3a). During the subsequent dissociation phase, the signal remained at a plateau, indicating no off-rate. The injection of rough LPS mutants and deep-rough LPS mutants (Figures 3b and 3c) showed similar binding curves except for a slight increase in the signal during the dissociation phase, while the purified polysaccharide did not show binding properties (Figure 3d).

[0167] These results indicated that all mutants containing the lipid A moiety strongly bound to GCN4-PII, while the pure polysaccharide did not, thus localizing the interaction to the lipid A moiety. However, the absence of an off-rate and the tendency of LPS to form aggregates in solution (Sasaki and White, 2008; Richter et al., 2011) meant that the characterization of the biophysical properties of this interaction is potentially complex, and these results could only be interpreted qualitatively. The increase in signal after injection of rough and deep rough mutants of LPS was thought to be inversely proportional to the number of sugar residues present in each mutant. In particular, deep rough LPS had a significantly higher hydrophobic-to-hydrophilic ratio and was a larger, less fluid form compared to LPS with longer sugar moieties (Richter et al., 2011). Therefore, the increase in signal after injection was interpreted as being due to the slower reorganization and degradation of deep rough aggregates compared to smooth mutants.

[0168] The construct was purified using a 6×His-tag, which implicitly demonstrated an endotoxin deficiency effect during purification due to nonspecific binding (Mack et al., 2014). To evaluate the effect of the His-tag on binding, two SadA constructs (K3 and K3-His) flanked by GCN4-pII, which were identical except for the His-tag, were compared. These constructs showed almost identical curves to each other and to the conventional construct, indicating that the His-tag does not affect binding (Figure 10).

[0169] We considered whether the nature of the interaction between GcN4-PII and LPS was hydrophobic, electrostatic, or a combination of both. The selection of the regeneration solution helped determine this. In the process of testing suitable regeneration buffers before the experiment, it was found that 1M NaCl was ineffective, but a mixture of undenatured surfactants up to 0.3% regenerated the sample in less than 60 seconds. This result indicated that a strong hydrophobic factor was involved in the interaction.

[0170] Example 2 – GCN4-PII binds with high affinity. SPR results were not suitable for determining the binding rate of the GCN4-pII / LPS interaction. To quantify affinity, we modified the previously described ELISA-like tail-spike adsorption (ELITA) assay (Schmidt et al., 2016) by using purified protein instead of whole bacteria. This assay was similar to the conventional ELISA except that the antibody was replaced with a phage tail-spike protein that recognizes the O antigen of LPS (Figure 11). The results showed that both constructs exhibited extremely high binding affinity in the low pM range, which is consistent with the no-off-rate observed in the SPR experiment (Figure 5). This construct proved advantageous as it allowed the use of LPS concentrations below the critical micelle concentration (CMC) of smooth LPS (Yu et al., 2006; Sasaki and White, 2008), which would have complicated interpretation if not modified. However, indirect ligand-receptor interaction constructs were not possible due to the tendency of LPS to coat the microtiter wells before blocking.

[0171] Example 3 – GCN4-pII dissolves LPS aggregates It was observed that adding GCN4-PII to LPS caused visible degradation of LPS aggregates. This phenomenon was investigated by comparing the structure of rough LPS at various GCN4-pII ratios using transmission electron microscopy (Figure 6). Prior to the experiment, it was confirmed that the synthesized GCN4-pII bound to LPS and maintained its α-helix structure using an LAL masking assay (Schwarz et al., 2017), circular dichroism, and NMR. The NMR spectrum confirmed that this peptide existed in a homologous α-helix state (Figure 14), that this state was maintained even after binding to LPS (Figure 12), and that at a GCN4-pII concentration of 1 μM, it exhibited at least an 89% neutralization effect (binding) to LPS (Figure 13).

[0172] Rough LPS was observed by TEM to form tubular micelles (Figure 6, top) with a radius of approximately 10 nm and a length ranging up to several hundred nm, as previously reported by cryo-EM (Richter et al., 2011; Broeker et al., 2018). After incubation with equimolar concentrations of GCN4-PII, the micelle structure completely disappeared, leaving behind occasional aggregates, likely caused by some aggregation of the peptide-LPS complex (Figure 6, bottom).

[0173] Discussion of Results (Examples 1-3) We attempted for the first time to investigate the presumed interaction between the trimer SadA domain and LPS. However, our results indicate that the GCN4-pII adapter used to stabilize our construct exhibits extremely high affinity for LPS. Interestingly, in the picomolar concentration range of K DThe affinity of GCN4-pII is 3 to 5 orders of magnitude higher than that of human LPS immunoreceptors TLR4 (141 μM), CD14 (74 nM), MD-2 (2.33 μM), and LPS-binding protein (3.5 nM). The dissociation constant obtained by the inventors with GCN4-pII is also 1 to 6 orders of magnitude higher than that of polymyxin B (48 μM) and peptide avibodies specifically designed for the highest possible affinity. Furthermore, in contrast to some of the aforementioned binding partners, the inventors have shown that GCN4-pII is specific to lipid A. The inventors have demonstrated using surfactants that this interaction is reversible and that GCN4-pII readily decomposes LPS aggregates in solution, showing that the interaction is largely hydrophobic. To the best of the inventors' knowledge, this application is the first report of a trimer-coiled-coil motif that binds to LPS. The previously reported crystal structure containing GCN4-pII (Hartmann et al., 2012) shows that the γ2 and δ-carbons belonging to the coisoleucine protrude from the core, forming a hydrophobic surface along the coiled-coil grooves. A model can be conceived that explains how GCN4-pII can degrade LPS aggregates, in which one or more lipid A acyl chains can align along these grooves to form extremely strong interactions. However, GCN4-pII also has a C-terminal patch of cationic residues, which may also contribute to the interactions.

[0174] Example 4 – Sensitivity of ELISA using GCN4-pII The purpose of this example was, in principle, to demonstrate that the binding of an oligomeric protein to LPS, as indicated by GCN4-pII in this application, can detect the amount of LPS with the same sensitivity as the LAL assay. As in Examples 1-3, SadA-utilizing constructs, particularly the K9 construct described above, were used.

[0175] To ensure complete reproducibility of this assay, sensitivity experiments were performed in four replicates using the optimized final conditions. Only the internally randomized wells were used to cancel out edge effects. The only exception was A3:A10, which was reserved for the highest concentration sample. For comparison, the same sample was subsequently measured using the LAL assay. Only one replicate was included in the results.

[0176] In a GCN4-pII-based ELISA (Figure 15) used to detect biotinylated LPS (B-LPS), a linear signal response was observed within the LPS concentration range of 0.06–1 ng / mL. This means that this assay can consistently detect B-LPS down to the lowest dilution concentration (0.06 ng / mL).

[0177] For comparison, a LAL assay was also performed. The concentration range of LPS used in the LAL assay was 0.01–0.1 EU / mL. The lowest concentration dilution that gave a clear signal was 0.13 ng / mL (Figure 16). This means that the GNC4-pII assay has a similar level of sensitivity to the LAL assay. A comparison of the results of these two assays is shown in Figure 17.

[0178] Example 5 – Strength of binding between GCN4-pII and LPS To investigate the binding strength between GCN4-pII and various LPS types, SPRs were used to check for a broad selection of LPS-mutants collected from various pathogens and proteobacteria (Table 4). In summary, K9 was immobilized on a carboxyl matrix on an SPR tip using EDC-NHS-assisted amine coupling. Various LPS types were injected three times in 0.5 mg / mL increments to observe the signal.

[0179] [Table 5]

[0180] In previous studies, the inventors compared the binding of LPS supplied from γ-proteobacteria, namely S. enterica, S. anatum, and Escherichia coli BL21. Injection of LPS yielded an immediate response, which approached a steady state towards the end of injection. During the subsequent dissociation phase, the signal remained plateaued, indicating no measurable off-rate. Although the shapes of the various curves were very similar, the final response (μRIU) changed inversely with each other in terms of the amount of sugar moiety per LPS molecule. This means that rough LPS types (lacking the O antigen) typically yielded a significantly stronger signal compared to their smooth counterparts (with O antigen repeats). Since all LPS types were injected at the same weight concentration (mg / mL), the differences in response likely reflected lower molar concentrations of the high molecular weight variants.

[0181] All LPS-type binding curves (Figure 18) checked by the inventors in their investigation of the present invention share similar binding curves with no off-velocity, indicating strong binding. S. tiphimulium WaaL LPS is a rough type and is expected to give a moderately high response when injected. N. lactamica and B. henselae are both large rough variants and were expected to give responses in the same range as WaaL, although N. lactamica gave a response that was almost twice as large, which can be explained by the large amount of sialic acid modification to the core sugar. V. cholerae has a mixture of rough LPS and low molecular weight smooth LPS. Therefore, the response of about one-third the WaaL signal was unexpected. The response of P. gingivalis LPS was low, suggesting a medium to large smooth LPS type. The documentation accompanying commercially available P. gingivalis LPS does not list the name of the strain or variant, so the weight must be determined by SDS-PAGE.

[0182] In conclusion, GCN4-pII bound to all LPS types tested.

[0183] Discussion of Results (Examples 4 and 5) The LAL assay utilizes an enzyme cascade in horseshoe crab blood that is highly sensitive to small amounts of LPS (Lee, 2007). Using the LAL assay in direct comparison, the inventors were able to demonstrate that the GCN4-pII peptide can bind to biotinylated LPS at concentrations barely detectable by the LAL assay, and that this binding still produces a visible signal when used with routine detection methods for biotin coupled with a fluorescent enzyme substrate. Importantly, this detection method worked in various buffer backgrounds and in injectable drug backgrounds. The present invention achieves a sensitivity of 0.01 EU / mL LPS comparable to the LAL assay, and the data from the present invention suggest that even higher sensitivity can be achieved in the ELISA-like assay of the present invention by, for example, fine-tuning the wash buffer conditions.

[0184] This invention utilizes various lineages of proteobacteria ranging from α-proteobacteria to γ-proteobacteria, as well as LPS mutants derived from the Bacteroidetes phylum (Figure 19). The selected bacteria of this invention include enteric pathogens (Vibrio cholerae, Salmonella, Escherichia coli), intracellular pathogens (Bartonella henselae), oral pathogens (Porphyromonas gingivalis), and symbiotic bacteria (Neisseria lactamica). One of the species used is known to have an LPS mutant that does not induce a strong immune response (Bartonella henselae) (Zaehringer et al., 2004).

[0185] In the tree shown in Figure 19, the genus Salmonella is placed together with the genus Escherichia and is not shown separately. In this figure, the genus Bartonella is closest to the shown Brucella, and Porphyromonas gingivalis is part of Bacteroides. Furthermore, the large groups of the phyla Firmicutes, Actinobacteria, and Spirochaetes do not possess LPS as part of their membrane components.

[0186] In summary, using SPR, the present invention was able to demonstrate that all LPS mutants used strongly bind to the GCN4-pII peptide. While there were visible differences in the "on-rate" of binding for each LPS mutant, there was no detectable "off-rate," suggesting that this peptide can detect all of these mutants similarly (albeit with slight differences in binding rate).

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Claims

1. The use of an oligomeric protein as a binder for binding to lipopolysaccharide (LPS), wherein the oligomeric protein has a coiled-coil structure comprising at least two monomer peptides, each monomer peptide may be the same or different, and can form an α-helix, and comprises at least one core sequence having at least 90% sequence identity with the heptad (7 amino acid) motif repeat sequence of SEQ ID NO:

1.

2. The core sequence comprises at least three heptad motifs a-b-c-d-e-f-g or variants thereof, each variant comprising the insertion or deletion of no more than one amino acid residue in the heptad motif. The use according to claim 1, wherein each of the letters a, b, c, d, e, f, and g represents positions 1 to 7 of the seven amino acid residues in the heptad motif.

3. The use according to claim 2, wherein at least 50% of the amino acid residues corresponding to positions a and d, which are positions 1 and 4, respectively, of the heptad motif or its variants included in the core sequence are hydrophobic residues.

4. The use according to any one of claims 1 to 3, wherein the core array is in contact with the flanking array on one or both sides.

5. The use according to claim 4, wherein the flanking sequence comprises one or more heptad motifs and / or one or more portions thereof.

6. The use according to claim 5, wherein the heptad motif in the flanking sequence corresponds to a heptad motif found in SEQ ID NO: 1 or a sequence having at least 90% sequence identity therewith, wherein at least one of the amino acid residues corresponding to positions a and d, which are positions 1 and 4, respectively, of the heptad motif in the flanking sequence, is a hydrophobic residue.

7. The use according to any one of claims 4 to 6, wherein the flanking sequence includes SEQ ID NO: 1, a part thereof, or a sequence having at least 90% sequence identity therewith, and at least 50% of the amino acid residues corresponding to positions a and d, which are positions 1 and 4, respectively, of the heptad motif found in SEQ ID NO: 1, a part thereof, or a sequence having at least 90% sequence identity therewith, are hydrophobic residues.

8. The use according to any one of claims 4 to 7, wherein the flanking sequence includes one or more linker sequences.

9. The use according to any one of claims 1 to 8, wherein each monomer peptide comprises two or more core sequences, the core sequences may be the same or different.

10. The use according to any one of claims 1 to 9, wherein the oligomer protein is a dimer, trimer, or tetramer.

11. The use according to any one of claims 1 to 10, wherein the oligomeric protein is a trimer.

12. The use according to any one of claims 1 to 11, wherein the monomer peptide is provided as separate chains.

13. The monomer peptides are linked together, as described in any one of claims 1 to 12.

14. The use according to claim 13, wherein the monomer peptides are linked together to form a single chain, or the monomer peptides are linked by one or more chemical crosslinks.

15. The use according to any one of claims 2 to 14, wherein each hydrophobic residue in the heptad motif or its variants is independently selected from the group consisting of leucine, isoleucine, valine, alanine, methionine and chemical derivatives thereof.

16. The use according to claim 15, wherein each hydrophobic residue is independently selected from leucine and isoleucine or chemical derivatives thereof.

17. The use according to claim 16, wherein the chemical derivative is fluoroleucine or fluoroisoleucine.

18. The use according to any one of claims 2 to 17, wherein at least 50% of the hydrophobic residues in the heptad motif or its variants are isoleucine or fluoroisoleucine.

19. (i) At least 50% of the amino acid residues corresponding to positions b, c, e, f, and g, which are positions 2, 3, 5, 6, and 7, respectively, in the heptad motif or its variants are polar residues; and / or (ii) The use according to any one of claims 2 to 18, wherein at least 5% of the amino acid residues corresponding to positions b, c, e, f, and g, which are positions 2, 3, 5, 6, and 7 in the heptad motif or variant thereof, are aliphatic residues.

20. The use according to any one of claims 1 to 19, wherein each monomer peptide comprises 18 to 40 amino acids.

21. The use according to any one of claims 1 to 20, wherein each monomer peptide contains at least four cationic amino acids within the core sequence.

22. The aforementioned oligomer protein has a size range of less than nanomolar concentration of K D The use according to any one of claims 1 to 21, wherein it binds to LPS.

23. The aforementioned oligomeric protein is (i) In the form of a combination or fusion with one or more additional components; (ii) fixed on a solid substrate; or (iii) The use according to any one of claims 1 to 22, wherein it is joined to a detection portion that can be directly detected.

24. (i) The protein is conjugated to a detection portion, an oligomerization portion, or an immobilization portion, or is in the form of a fusion protein with a fusion partner; (ii) The protein is immobilized on beads or resin, or in or on wells, containers, columns or filter materials, or on the surface of a detection device; or, (iii) The use according to claim 23, wherein the detection portion is a spectrophotometrically detectable or spectroscopically detectable marker.

25. The use of the oligomeric protein as described in any one of claims 1 to 24, comprising the detection and / or removal of LPS in or from the sample.

26. A method for binding to LPS, comprising the step of contacting the LPS or a sample containing the LPS with an oligomeric protein as defined in any one of claims 1 to 24, thereby enabling the protein to bind to the LPS so as to form a protein-lipopolysaccharide complex.

27. The method further includes detecting the presence of LPS in the sample, and the method is (a) The step of contacting the sample with an oligomeric protein as defined in any one of claims 1 to 24, thereby enabling the protein to bind to the LPS so as to form a protein-lipopolysaccharide complex; (b) The method according to claim 26, comprising the step of detecting the presence of a protein-lipopolysaccharide complex.

28. The method further includes removing LPS from the sample, and the method is (a) The step of contacting the sample with an oligomeric protein as defined in any one of claims 1 to 24, thereby enabling the protein to bind to the LPS so as to form a protein-lipopolysaccharide complex; (b) The method according to claim 26, comprising the step of separating the protein-lipopolysaccharide complex from the sample.

29. The method according to any one of claims 26 to 28, wherein the oligomeric protein is in the form of a conjugate containing a detectable label, and / or the oligomeric protein is immobilized on a solid substrate.

30. The method according to any one of claims 26 to 29, wherein the sample is a clinical sample derived from a patient or a sample of a product for testing for endotoxin contamination.

31. The method according to claim 30, wherein the sample is a blood sample or a sample derived from a blood sample.

32. (i) an oligomeric protein as defined in any one of claims 1 to 18; (ii) A kit comprising at least one non-denaturing surfactant.

33. The kit according to claim 32, wherein the kit is for use in any one of claims 1 to 25 or in the method according to any one of claims 26 to 31.

34. A product comprising an oligomeric protein immobilized on a solid substrate, wherein the oligomeric protein is as defined in any one of claims 1 to 22.

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