High-affinity purification of ferritin

Abstract

The present invention relates to a novel protein that specifically binds to ferritin. The novel protein of the present invention is an advanced and powerful tool, since it allows a method for precise purification of ferritin, for example by affinity chromatography. Furthermore, the ferritin-binding protein is useful in a method for analyzing the presence or absence of ferritin.

Description

Technical Field

[0001] The present invention relates to a novel protein that specifically binds to ferritin. The novel protein of the present invention enables a method for the precise purification of ferritin, for example, by affinity chromatography, and is thus a highly powerful tool. Furthermore, the ferritin-binding protein is useful for a method for analyzing the presence or absence of ferritin.

Background Art

[0002] Ferritin is a large, multi-subunit protein complex and is the major iron storage protein in cells. Iron is an essential nutrient required for oxygen transport in the bloodstream and as a component of enzymes for oxidative respiration. Iron deficiency has a significant impact on, for example, the neurological development of children.

[0003] Iron storage and transport proteins are located mainly in the liver, spleen, and bone marrow, but also in serum and plasma. This protein is composed of subunits of four-helix bundle proteins arranged in a 24-mer with strict symmetry.

[0004] Purified ferritin can be administered to individuals who require iron and can be used to treat or supplement iron deficiency disorders.

[0005] Furthermore, ferritin has also been described as part of a drug delivery vehicle for tumor-targeted therapy.

[0006] Therefore, there is a strong need in the art to provide an efficient method for purifying ferritin. The present invention satisfies this need by providing a particularly advantageous novel ferritin-binding protein that enables the precise purification of ferritin, especially by affinity chromatography, for further use in medical applications.

[0007] The above summary does not necessarily describe all the problems solved by the present invention. [Overview of the project]

[0008] This disclosure provides, but is not limited to, the following items 1-15: 1. A ferritin-binding protein comprising an amino acid sequence having at least 90% sequence identity with one selected from the group of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, wherein the binding protein has a binding affinity of less than 100 nM to ferritin. 2.2, 3, 4, 5, or 6 binding proteins linked to each other, as described in item 1. 3. The binding proteins described in items 1-2, wherein this binding protein is fused to at least one non-ferritin-binding protein. 4. A fusion protein comprising at least one binding protein and at least one non-ferritin binding protein as described in claim 1. 5. The fusion protein described in item 4, comprising an attachment site for site-specific binding to a solid support. 6. A ferritin-binding protein as described in any one of items 1-3 or a fusion protein as described in items 4-5, for use in technologies such as affinity chromatography. 7. An affinity isolation matrix containing a ferritin-binding protein as described in any one of items 1-3 or a fusion protein as described in items 4-5. 8. Use of a ferritin-binding protein as described in any one of items 1-3, a fusion protein as described in items 4-5, or an affinity separation matrix as described in item 8 for affinity chromatography. 9. A method for purifying ferritin, wherein this method is (i) To provide a liquid containing ferritin, (ii) To provide the affinity isolation matrix comprising at least one ferritin-binding protein described in any one of items 1 to 3 or a fusion protein described in items 4 to 5, bound to the affinity isolation matrix. (iii) Contacting the affinity separation matrix with the liquid under conditions that enable the binding of at least one ferritin-binding protein described in any one of items 1 to 3 or a fusion protein described in items 4 to 5, and (iv) A method comprising eluting the ferritin from the affinity purification matrix. 10. Use of ferritin-binding proteins described in items 1-3, fusion proteins described in items 4-5, or affinity matrices described in item 7 in a method for analyzing the presence or absence of ferritin. 11. A method for analyzing the presence or absence of ferritin in a liquid sample, the method comprising the following steps: (i) A step of providing a liquid containing ferritin, (ii) A step of providing a ferritin-binding protein as described in items 1 to 3 or a fusion protein as described in items 4 to 5, (iii) The step of bringing the liquid of (i) into contact with the ferritin-binding protein described in items 1-3 or the fusion protein described in items 4-5 under conditions that enable binding of the ferritin-binding protein, (iv) The step of isolating the ferritin and binding protein complex, (v) The step of determining the amount of ferritin-binding protein in the liquid of (i), method. 12. A nucleic acid molecule encoding a ferritin-binding protein as described in items 1-3 or a fusion protein as described in items 4-5. 13. A vector containing the nuclear molecule described in item 12. 14. Host cells or non-human hosts containing ferritin-binding proteins as described in items 1-3 or fusion proteins as described in items 4-5, nucleic acids as described in item 12, and / or vectors as described in item 13. 15. A method for producing a ferritin-binding protein according to items 1-3 or a fusion protein according to items 4-5, comprising culturing the host cells of item 14 under appropriate conditions to obtain the ferritin-binding protein or a fusion protein containing the ferritin-binding protein, and optionally isolating the ferritin-binding protein or a fusion protein containing the ferritin-binding protein.

[0009] This summary of the present invention is not limiting, and other aspects and embodiments of the present invention will become apparent from the following description, examples, and drawings. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 shows the amino acid sequences of ferritin-binding proteins (SEQ ID NOs: 2, 3, and 4). Identical or similar amino acids are labeled with an asterisk (*) and a double dot (:), respectively. Alignment was performed using a multi-sequence alignment tool. [Figure 2] Figure 2 shows the SPR of 219127 (a fusion protein including SEQ ID NO: 2) immobilized via the c-terminal cysteine ​​residue using PDEA binding. 219127 binds to ferritin with an affinity of less than 0.5 nM even at pH 4.5. [Figure 3]Figure 3 shows the purification of ferritin using an affinity ligand immobilized on Praesto85. Figure 3A shows the elution chromatogram of an affinity chromatography run performed with fusion protein 219127 bound to Praesto85 resin. The solid black line indicates the UV 280 nm signal. The gradient of the elution buffer is shown by the dashed black line. The figure shows a uniform elution profile of ferritin with a single peak. The maximum peak in the pH gradient profile was at pH 3.1. Figure 3B shows the SDS-PAGE of the analyzed fraction of the affinity chromatography run using Praesto85-219127. A liquid containing ferritin was loaded. Purified human ferritin is visible as the MW band 20 kDa (arrow). No significant impurities were detected. Lane 1: protein marker, Lane 2: ferritin, Lanes 3-7: pass-through fraction, Lane 8: wash fraction (washed at pH 4.5), Lanes 9-10: elution fraction. [Modes for carrying out the invention]

[0011] The present invention provides a novel protein having specific binding affinity to ferritin. The novel protein of the present invention is particularly advantageous as an affinity ligand for ferritin, for example, because it enables precise purification of ferritin in affinity chromatography. Any polypeptide selected from the group of SEQ ID NOs: 1, 2, 3, and 4, or an amino acid sequence having at least 90% identity with any one of SEQ ID NOs: 1, 2, 3, and 4, each binds to human ferritin with high affinity.

[0012] Before describing the present invention in more detail below, it should be understood that the specific methodologies, protocols, and reagents described herein may be modified and are not limited to them. It should also be understood that the terms used herein are for the purpose of describing specific aspects and embodiments only and are not intended to limit the scope of the present invention as shown in the accompanying documentation. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one person skilled in the art in which the present invention pertains. This includes those skilled in the art working in the fields of protein engineering and purification, but also those skilled in the art working in the field of developing novel specific ferritin-binding molecules for use in technical applications, such as for use as affinity ligands in affinity chromatography.

[0013] Preferably, the terms used herein are defined as those set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)," Leuenberger, HGW, Nagel, B., and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0014] Throughout the specification of this invention and the subsequent documents, unless the context requires otherwise, the words “comprise,” and variations such as “comprises,” and “comprising,” are understood to mean that a given integer or step, or group of integers or steps, is included, but not that any other integer or step, or group of integers or steps is excluded. The terms “comprise(s)” or “comprising” may include any limitation to “consists of” or “consisting of,” where such limitation is necessary for any reason or to any degree.

[0015] Throughout the specification of the present invention, several documents (such as patents, patent applications, scientific literature, manufacturer's specifications, instructions, UniProt Accession Number, etc.) may be cited. In this specification, the invention should not be construed as an admission that it has no right to precede such disclosure by a prior invention. Some of the documents cited in the present invention may be characterized as "incorporated by reference." If there is a conflict between the definition or teaching of such incorporated references and the definitions or teachings recited in this specification, the language of this specification shall prevail.

[0016] All sequences referred to in this specification are disclosed in the accompanying sequence listing, which forms part of the disclosure content of this specification by the content and the entire disclosure.

[0017] General definitions of important terms used in this specification The term "ferritin" refers to the amino acid sequence shown in UniProtKB Q8TD27. The term "ferritin" includes all polypeptides showing at least 70%, 80%, 85%, 90%, 95%, 96% or 97% or more, or 100% amino acid sequence identity with UniProtKB Q8TD27. In some embodiments, ferritin relates to mammalian ferritin. In some embodiments, ferritin relates to human ferritin.

[0018] The terms "binding protein for ferritin", "ferritin binding protein" or "affinity ligand" may be used interchangeably herein and describe a protein that can bind to ferritin. As described herein, a ferritin binding protein refers to a protein having a detectable interaction with ferritin, determined by an appropriate method such as SPR analysis or BLI or other appropriate techniques known to those skilled in the art.

[0019] The term "non-ferritin-binding protein" refers to a protein that does not have a detectable interaction with ferritin, as determined by appropriate methods such as SPR analysis, BLI, or other appropriate techniques known to those skilled in the art.

[0020] The term "binding affinity" refers to the ability of the polypeptide of the present invention to bind to ferritin. Binding affinity is typically used to evaluate and grade the strength of bimolecule interactions using the equilibrium dissociation constant (K). D ) are measured and reported. Binding affinity and dissociation constant can be measured quantitatively. Methods for measuring binding affinity are well known to those skilled in the art and can be selected from, for example, the following established methods in the art: surface plasmon resonance (SPR), biolayer interferometry (BLI), enzyme-linked immunosorbent assay (ELISA), binding equilibrium exclusion assay (KinExA assay), flow cytometry, fluorescence spectroscopy, isothermal titration calorimetry (ITC), analytical ultracentrifugation, radioimmunoassay (RIA or IRMA), and enhanced chemiluminescence (ECL). Typically, the dissociation constant K D Measurements are taken at temperatures in the range of 20°C to 30°C. Unless otherwise specified, the K values ​​listed herein are used. D This is determined by SPR at 25°C. In various embodiments of the present invention, the binding affinity to ferritin may be determined by the Sierra SPR-32 system (Bruker).

[0021] The term "fusion protein" refers to a protein that includes at least one first protein genetically linked to at least one second protein. Fusion proteins are created by linking two or more genes that originally encode separate proteins. Therefore, fusion proteins may contain a polymer of identical or different proteins that are expressed as a single linear polypeptide.

[0022] The term "amino acid sequence identity" refers to the quantitative comparison of the identity (or differences) of the amino acid sequences of two or more proteins. The "amino acid sequence identity percentage (%)" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a given sequence that are identical to those in the reference polypeptide sequence, after the sequences have been aligned and gaps introduced to achieve the maximum possible sequence identity percentage, if necessary. To determine sequence identity, the sequence of the query protein is aligned to a reference protein or polypeptide, for example, the polypeptide sequence of SEQ ID NO: 1. Sequence alignment methods are well known in the art. For example, to determine the degree of amino acid sequence identity of any polypeptide to, for example, the amino acid sequence of SEQ ID NO: 1, it is preferable to use the freely available SIM local similarity program (Xiaoquin Huang and Webb Miller (1991), Advances in Applied Mathematics, vol.12:337-357). For multiple sequence alignment, it is preferable to use ClustalW (Thompson et al. (1994) Nucleic Acids Res., 22(22):4673-4680).

[0023] The terms “protein” and “polypeptide” refer to two or more amino acids in any chain linked by peptide bonds, and do not refer to a product of a specific length. Therefore, any other term used to refer to “peptide,” “protein,” “amino acid chain,” or a chain of two or more amino acids is included within the definition of “polypeptide,” and the term “polypeptide” may be used in place of or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to post-translational modification products of polypeptides, such as glycosylation, which is well known in the art.

[0024] The term "alkaline stability" refers to the ability of a ferritin-binding protein to withstand alkaline conditions without significantly losing its ability to bind to ferritin. Those skilled in the art can easily investigate this by, for example, incubation a ferritin-binding protein in a sodium hydroxide solution as described in the examples, and then testing its binding affinity to ferritin by routine experiments known to those skilled in the art, such as chromatography.

[0025] The term "chromatography" refers to a separation technique that utilizes a mobile phase and a stationary phase to separate one type of molecule (e.g., ferritin) from other molecules in a sample. The liquid mobile phase contains a mixture of multiple types of molecules, which are moved across or through the stationary phase (e.g., a solid matrix). Due to the differences in the interactions between the different types of molecules in the mobile phase and the stationary phase, the molecules in the mobile phase can be separated.

[0026] The term "affinity chromatography" refers to a specific mode of chromatography in which a ligand (i.e., a ferritin-binding protein) bound to the stationary phase interacts with a molecule (i.e., ferritin) in the mobile phase (i.e., a liquid sample), i.e., the ligand has a specific binding affinity to the molecule that is captured. As understood in the context of this invention, affinity chromatography involves adding a ferritin-containing (liquid) sample to a stationary phase containing a chromatography ligand, such as a ferritin-binding protein. The terms "solid support" or "solid matrix" are used interchangeably for the stationary phase.

[0027] As used interchangeably herein, the terms “affinity matrix” or “affinity purification matrix” refer to a matrix, such as a chromatographic matrix on which affinity ligands (e.g., ferritin-binding proteins or fusion proteins containing ferritin-binding proteins) are attached. The attached affinity ligands can specifically bind to the target molecule (e.g., ferritin) that is to be generated or removed from the liquid. The liquid may be, but is not limited to, serum or crystals.

[0028] As used herein, the term "affinity purification" refers to a method of purifying (capturing) ferritin from a liquid by binding it to a ferritin ligand immobilized on a matrix. This removes the ferritin from the liquid.

[0029] Detailed description of embodiments of the present invention The present invention will be further described. The following section defines various aspects of the present invention in more detail. Each aspect defined below may be combined with any other aspect or combination of aspects unless otherwise expressly specified. In particular, any feature shown to be preferred or advantageous may be combined with any other feature or combination of features shown to be preferred or advantageous.

[0030] The novel ferritin-binding protein exhibits specific binding affinity to ferritin. The ferritin-binding protein contains an amino acid sequence selected from the group of SEQ ID NOs: 1, 2, 3, and 4, or an amino acid sequence having at least 90% sequence identity with one of SEQ ID NOs: 1, 2, 3, or 4.

[0031] In some embodiments, the ferritin-binding protein contains the amino acid sequence of SEQ ID NO: 1. In some embodiments, the ferritin-binding protein contains amino acids having at least 90% sequence identity with SEQ ID NO: 1.

[0032] In some embodiments, the ferritin-binding protein contains the amino acid sequence of SEQ ID NO: 2. In some embodiments, the ferritin-binding protein contains amino acids having at least 90% sequence identity with SEQ ID NO: 2.

[0033] In some embodiments, the ferritin-binding protein contains the amino acid sequence of SEQ ID NO: 3. In some embodiments, the ferritin-binding protein contains amino acids having at least 90% sequence identity with SEQ ID NO: 3.

[0034] In some embodiments, the ferritin-binding protein contains the amino acid sequence of SEQ ID NO: 4. In some embodiments, the ferritin-binding protein contains amino acids having at least 90% sequence identity with SEQ ID NO: 4.

[0035] In some embodiments, the ferritin-binding protein includes at least one amino acid sequence shown in Figure 1.

[0036] In some embodiments, the ferritin-binding protein has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 1-4.

[0037] In some embodiments, the amino acids corresponding to positions 1-4, 7, 9-11, 14, 15, 17, 19-40, 42, 43, 45-47, 49-51, and 53-56 of the ferritin-binding protein shown in the amino acid sequences of SEQ ID NOs: 1-4 are identical.

[0038] The amino acid corresponding to position 5 in Sequence ID No. 1 may be selected from Q or A.

[0039] The amino acid corresponding to position 6 in sequence number 1 may be selected from aromatic amino acids (W, Y, F).

[0040] The amino acid corresponding to position 8 of SEQ ID NO: 1 may be selected from S or L, and is preferably S.

[0041] The amino acid corresponding to position 12 in SEQ ID NO: 1 may be selected from either K or H.

[0042] The amino acid corresponding to position 13 in sequence number 1 may be selected from E or Q.

[0043] The amino acid corresponding to position 16 of SEQ ID NO: 1 may be selected from K, E, or Q. The amino acid corresponding to position 18 of SEQ ID NO: 1 may be selected from P or F, preferably P.

[0044] The amino acid corresponding to position 41 of sequence number 1 may be selected from E or Y, and is preferably E.

[0045] The amino acid corresponding to position 44 in SEQ ID NO: 1 may be selected from W or Q.

[0046] The amino acid corresponding to position 48 of SEQ ID NO: 1 may be selected from E, V, or M. The amino acid corresponding to position 52 of SEQ ID NO: 1 may be selected from S or A, preferably S.

[0047] One embodiment refers to a ferritin-binding protein comprising an amino acid sequence having at least 90% sequence identity with any one selected from the group of SEQ ID NOs: 2, 3, or 4.

[0048] One embodiment refers to a ferritin-binding protein comprising an amino acid sequence having at least 90% sequence identity with any one selected from the group of SEQ ID NOs: 2 and SEQ ID NOs: 3.

[0049] One embodiment refers to a ferritin-binding protein comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2.

[0050] In some embodiments, the ferritin-binding protein has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 3, and the amino acids corresponding to positions 1-4, 7-11, 14, 15, 17-43, 45-47, and 49-56 of SEQ ID NO: 2 or SEQ ID NO: 3 are not modified.

[0051] In some embodiments, the ferritin-binding protein has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 3, and one or more amino acids corresponding to positions 5, 6, 8, 12, 13, 16, 44, and 48 of SEQ ID NO: 2 or SEQ ID NO: 3 are modified.

[0052] One advantage of the ferritin-binding proteins disclosed herein is their important functional property of specifically binding to ferritin with low affinity. Needless to say, this is particularly advantageous when purifying ferritin in protein purification processes. The ferritin-binding proteins are functionally characterized by a binding affinity of less than 100 nM to ferritin. In some embodiments, the ferritin-binding proteins are functionally characterized by a binding affinity of less than 50 nM to ferritin. In some embodiments, the ferritin-binding proteins are functionally characterized by a binding affinity of less than 10 nM to ferritin.

[0053] Multimer. In one embodiment of the present invention, the ferritin-binding protein comprises one, two, three, four, five, or six linked binding proteins. In one embodiment of the present invention, the ferritin-binding protein comprises one ferritin-binding protein or two linked ferritin-binding proteins. Multimers of binding proteins are generally produced artificially by recombinant DNA techniques well known to those skilled in the art. In some embodiments, the multimer is a homomultimer, for example, having identical amino acid sequences of the ferritin-binding proteins. In other embodiments, the multimer is a heteromultimer, for example, having different amino acid sequences of the ferritin-binding proteins.

[0054] Fusion protein. In some embodiments, the ferritin-binding protein described above is fused with one further polypeptide different from the disclosed polypeptide. In various embodiments, the further polypeptide different from the ferritin-binding protein disclosed herein may be a non-ferritin-binding protein. In some embodiments, the further non-ferritin-binding polypeptide is a non-immunoglobulin (Ig)-binding protein, such as, for example, a protein that does not bind to Ig. In some embodiments, a common structural feature of the non-ferritin-binding protein is that the protein has a triple-helical protein A-like structure comparable to the structure of the ferritin-binding protein disclosed herein.

[0055] In some embodiments, the non-ferritin-binding protein has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO: 11.

[0056] Some embodiments include a fusion protein comprising a ferritin-binding protein and at least one non-ferritin-binding polypeptide disclosed herein.

[0057] Some embodiments include fusion proteins comprising a ferritin-binding protein and one or more non-ferritin-binding polypeptides disclosed herein.

[0058] In some embodiments, a fusion protein comprising at least one ferritin-binding protein and at least one non-ferritin-binding protein selected from SEQ ID NOs: 1-4.

[0059] In some embodiments, a fusion protein comprising at least one ferritin-binding protein selected from SEQ ID NOs: 1 to 4 and at least one non-ferritin-binding protein having at least 89% identity with SEQ ID NO: 11.

[0060] In some embodiments, the fusion protein comprises a ferritin-binding protein, the C-terminus of which is fused to the N-terminus of a non-ferritin-binding protein. In some embodiments, the fusion protein comprises at least one ferritin-binding protein, the C-terminus of which is fused to the N-terminus of a dimer of a non-ferritin-binding protein having at least 89% amino acid sequence identity with SEQ ID NO: 11 or at least 90% amino acid sequence identity with SEQ ID NO: 11.

[0061] In one embodiment, the fusion protein includes SEQ ID NO: 2 fused to two non-ferritin-binding proteins (see, for example, SEQ ID NO: 5 (219126)).

[0062] In one embodiment, the fusion protein comprises a dimer of SEQ ID NO: 2 fused to two non-ferritin-binding proteins (see, for example, SEQ ID NO: 6 (219127)).

[0063] In one embodiment, the fusion protein includes SEQ ID NO: 3 fused to two non-ferritin-binding proteins (see, for example, SEQ ID NO: 7 (219124)).

[0064] In one embodiment, the fusion protein comprises a dimer of SEQ ID NO: 3 fused to two non-ferritin-binding proteins (see, for example, SEQ ID NO: 8 (219125)).

[0065] In one embodiment, the fusion protein includes SEQ ID NO: 4 fused to two non-ferritin-binding proteins (see, for example, SEQ ID NO: 9 (219122)).

[0066] In one embodiment, the fusion protein comprises a dimer of SEQ ID NO: 4 fused to two non-ferritin-binding proteins (see, for example, SEQ ID NO: 10(219123)).

[0067] In some embodiments, the fusion protein includes attachment sites for site-specific binding to a solid support, as further described below.

[0068] Molecules for purification or detection. In some embodiments, the ferritin-binding protein may also include additional amino acid residues at the N and / or C terminus, such as additional sequences at the N and / or C terminus. The additional sequences may include, for example, sequences introduced for purification or detection. Typical examples of such sequences include, but are not limited to, Strep-tag, oligohistidine-tag, glutathione S-transferase, maltose-binding protein, intein, intein fragment, or the albumin-binding domain of protein G, or others. In one embodiment, the additional amino acid sequence includes one or more peptide sequences that provide affinity to a particular chromatography column material. The ferritin-binding protein or fusion protein containing the ferritin-binding protein may preferably include a specific attachment site at the C terminus for attachment to a solid support such as cysteine ​​or lysine.

[0069] Use of novel ferritin-binding proteins in technical applications. This specification also provides the use of any novel ferritin-binding proteins disclosed herein, including fusion proteins, in technical applications, preferably for use in affinity purification.

[0070] Affinity purification of ferritin. As described herein, affinity chromatography (also known as affinity purification) utilizes specific intermolecular binding interactions. Methods for protein immobilization and affinity chromatography are well known in the field of protein purification and can be easily performed by those skilled in the art using standard techniques and equipment.

[0071] Some embodiments refer to a method for purifying ferritin, the method comprising: (i) providing a liquid containing ferritin; (ii) providing this affinity isolation matrix comprising the at least one ferritin-binding protein or the fusion protein described above bound to the affinity isolation matrix; (iii) contacting the affinity isolation matrix with the liquid under conditions that enable the binding of the at least one ferritin-binding protein or the fusion protein described above; and (iv) eluting the ferritin from the affinity purification matrix.

[0072] Some embodiments refer to a method for purifying ferritin, the method comprising: (i) providing a liquid containing ferritin, which is plasma or serum, or obtained from plasma or serum; (ii) providing an affinity isolation matrix comprising the above-mentioned at least one ferritin-binding protein or the above-mentioned fusion protein bound to the affinity isolation matrix; (iii) contacting the affinity isolation matrix with the liquid under conditions that enable the binding of the above-mentioned at least one ferritin-binding protein or the above-mentioned fusion protein; and (iv) eluting the ferritin from the affinity purification matrix.

[0073] In various embodiments, the affinity purification method may further include one or more washing steps. Affinity purification matrices suitable for the disclosed uses and methods are known to those skilled in the art.

[0074] In various embodiments, the affinity purification method may further include one or more steps of isolating ferritin proteins purified by methods known to those skilled in the art.

[0075] Binding to a Solid Support. In various aspects and / or embodiments of the present invention, the novel ferritin-binding proteins disclosed herein, including novel ferritin-binding proteins prepared or obtained by any of the methods described above, are bound to a solid support. In some embodiments, the ferritin-binding protein includes attachment sites for site-specific covalent binding of the ferritin-binding protein to a solid support. Examples of specific attachment sites include, but are not limited to, native amino acids such as cysteine ​​or lysine, which enable specific chemical reactions with reactive groups of the solid phase or linkers between the solid phase and the protein.

[0076] Affinity purification matrix. In another embodiment, an affinity purification matrix is ​​provided that includes a ferritin-binding protein or a fusion protein comprising a ferritin-binding protein.

[0077] In preferred embodiments, the affinity purification matrix is ​​a solid support. The affinity purification matrix comprises at least one ferritin-binding protein or a fusion protein comprising a ferritin-binding protein as described herein. Thus, novel ferritin-binding proteins or fusion proteins comprising ferritin-binding proteins disclosed herein are included in the use of affinity purification matrices in the purification of ferritin.

[0078] Solid support matrices for affinity chromatography are well known in the art and include, but are not limited to, agarose and stabilized agarose derivatives, cellulose or cellulose derivatives, controlled pore glass, monoliths, silica, zirconium oxide, titanium oxide, or synthetic polymers, and hydrogels of various compositions, as well as combinations thereof.

[0079] The form of the solid support matrix may be any suitable and well-known type. Such a solid support matrix for binding to the novel proteins or polypeptides of the present invention may include, but are not limited to, one of the following: columns, capillaries, particles, membranes, filters, monoliths, fibers, pads, gels, slides, plates, cassettes, or any other form commonly used in chromatography and known to those skilled in the art.

[0080] In one embodiment, the matrix consists of substantially spherical beads, such as Sepharose or agarose beads. The matrix in particle form can be used as a packed bed or in a suspension form including an expanded bed. In other embodiments of the present invention, the solid support matrix is ​​a membrane, such as a hydrogel membrane. In some embodiments, affinity purification may include a membrane as a matrix to which the ferritin-binding proteins described herein are covalently bound. The solid support may also be in the form of a membrane in a cartridge.

[0081] In some embodiments, affinity purification includes a chromatography column containing a solid support matrix to which the novel proteins of the present invention are covalently bound. The ferritin-binding proteins or fusion proteins containing the ferritin-binding proteins described above may be attached to a suitable solid support matrix by conventional binding techniques. Methods for immobilizing protein ligands on solid supports are well known in the fields of protein engineering and purification and can be easily carried out by those skilled in the art using standard techniques and equipment.

[0082] Uses and methods for determining the presence or absence of ferritin. In some embodiments, the use of the ferritin-binding protein, the fusion protein, or the affinity matrix described above in a method for determining the presence or absence of ferritin is referred to.

[0083] In some embodiments, the ferritin-binding proteins or fusion proteins described herein are used in a method for determining the presence or absence of ferritin. In some embodiments, a method for analyzing the presence or absence of ferritin in a liquid sample is described, comprising the steps of: (i) providing a liquid containing ferritin; (ii) providing a ferritin-binding protein; (iii) contacting the liquid containing ferritin with the ferritin-binding protein or fusion protein containing a ferritin-binding protein described herein under conditions that enable the binding of at least one ferritin-binding protein to ferritin; (iv) isolating (eluting) a complex of ferritin and the ferritin-binding protein or fusion protein containing a ferritin-binding protein; and (v) determining the amount of ferritin-binding protein indicating the amount of ferritin in the liquid of (i).

[0084] A method for quantifying ferritin. Further embodiments relate to a method for quantifying ferritin, comprising: (i) providing a liquid containing ferritin; (ii) providing a matrix on which ferritin-binding proteins or fusion proteins containing ferritin-binding proteins as described herein are covalently bound; (iii) contacting the affinity-purified matrix with the liquid under conditions that enable the binding of at least one ferritin-binding protein or fusion protein containing at least one ferritin-binding protein to ferritin; (iv) eluting the ferritin; and (v) quantifying the amount of eluted ferritin. The method for determining the presence or absence of ferritin in a liquid sample may be quantitative or qualitative. Such methods are well known to those skilled in the art and can be selected from, for example, the following established methods: enzyme-linked immunosorbent assay (ELISA), enzymatic reactions, surface plasmon resonance (SPR), or chromatography.

[0085] Polynucleotides, vectors, and host cells. One embodiment comprises an isolated polynucleotide or nucleic acid molecule encoding a ferritin-binding protein or a fusion protein containing a ferritin-binding protein as described herein. Further embodiments also encompass proteins encoded by polynucleotides.

[0086] Furthermore, this specification provides vectors, particularly expression vectors, comprising isolated polynucleotides or nucleic acid molecules of the ferritin-binding protein or fusion protein containing the ferritin-binding protein described herein, as well as host cells comprising the isolated polynucleotides or expression vectors. For example, one or more polynucleotides encoding the ferritin-binding protein or fusion protein containing the ferritin-binding protein disclosed herein may be expressed in a suitable host, and the resulting protein can be isolated. A vector means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) that can be used to transmit protein-coding information to a host cell. Suitable vectors that may be applied are known in the art.

[0087] Suitable host cells include prokaryotes or eukaryotes, such as vector-bearing bacterial host cells, yeast host cells, or non-human host cells. A suitable host may also be a microbial expression host capable of expressing the protein of the present invention. Suitable bacterial expression host cells or systems are known in the art. Various mammalian or insect cell culture systems known in the art can also be used to express recombinant proteins.

[0088] Furthermore, the Specified herein provides isolated cells containing polynucleotides or nucleic acids, or vectors.

[0089] A method for producing the protein of the present invention. Further embodiments provide a method for producing the ferritin-binding protein or a fusion protein comprising the ferritin-binding protein described herein, the method comprising (i) culturing (appropriate) host cells under conditions appropriate for expressing the ferritin-binding protein or the fusion protein comprising the ferritin-binding protein in order to obtain the ferritin-binding protein or the fusion protein comprising the ferritin-binding protein, and (ii) optionally isolating the ferritin-binding protein or the fusion protein comprising the ferritin-binding protein. Appropriate conditions for culturing prokaryotic or eukaryotic hosts are well known to those skilled in the art.

[0090] Ferritin-binding proteins or fusion proteins containing ferritin-binding proteins may be prepared by any conventional and well-known techniques, such as simple organic synthesis methods or solid-phase assisted synthesis techniques, or by commercially available automated synthesis equipment. These proteins may also be prepared using conventional recombination techniques, either alone or in combination with conventional synthesis techniques.

[0091] In one embodiment, as detailed above, a method is provided for preparing a ferritin-binding protein or a fusion protein comprising a ferritin-binding protein, the method comprising: (i) providing a nucleic acid molecule encoding a ferritin-binding protein or a fusion protein comprising a ferritin-binding protein; (ii) introducing this nucleic acid molecule into an expression vector; (iii) introducing this expression vector into host cells; (iv) culturing the host cells in a culture medium; (v) subjecting the host cells to culture conditions suitable for expression, thereby producing a ferritin-binding protein or a fusion protein comprising a ferritin-binding protein; optionally (vi) isolating the polypeptide produced in step (v); and (vii) optionally binding the ferritin-binding protein or the fusion protein comprising a ferritin-binding protein to the solid support described above. In various embodiments of the present invention, the production of a ferritin-binding protein or a fusion protein comprising a ferritin-binding protein is carried out by cell-free in vitro transcription and translation.

[0092] The following embodiments are provided for further explanation of the present invention. However, the present invention is not limited thereto, and the following embodiments merely demonstrate the feasibility of the present invention based on the above description. [Examples]

[0093] For further explanation of the present invention, the following embodiments are provided. However, the present invention is not limited thereto, and the following embodiments merely demonstrate the feasibility of the present invention based on the above description. For example, for a complete disclosure of the present invention, also refer to the documents cited herein, which are fully incorporated herein by reference.

[0094] Example 1. Selection and Screening Libraries. Dedicated cDNA libraries based on stable protein A-like variants (artificial mosaic proteins composed of protein A domain fragments and additional mutations) were synthesized in-house using randomized oligonucleotides with synthetic trinucleotide phosphoramidites (ELLA Biotech) or externally using Geneart, achieving a balanced amino acid distribution by simultaneously excluding cysteine ​​and other amino acid residues at randomization sites. In the following selection process using phage display, the corresponding cDNA libraries were amplified by PCR and ligated to pCD33-OmpA phagemides. Aliquots of the ligated mixture were used to electroporate E. coli SS320 (E. coli SS320) to prepare and purify phage libraries, which were then stored as cold storage.

[0095] Selection by phage display. Unless otherwise specified, established recombinant genetic methods were used. Naive libraries were enriched for human ferritin (as directed by Calbiochem, CAS 9007-73-2) as the ON target using phage display as the selection system. In each round, a selection step was performed using empty magnetic Dynabeads® M-270 Epoxy blocked with a Sigmablocker. The AIT method was applied, meaning that the ON target protein was immobilized on magnetic Dynabeads® M-270 Epoxy before starting each round. E. coli SS320 (Lucigene) was used for infection with the cold phage library, and E. coli ER2738 (Lucigene) was used for re-amplification of the phage pool after each round. Phage amplification and purification were performed using standard methods known to those skilled in the art. All three selection rounds were performed using an automated KingFisher system (Thermo Fisher) to isolate and capture the desired phage target complex. The target concentration started at 40 nM (round 1) and decreased to 10 nM in each round (round 3). The bound phages were eluted with trypsin and re-amplified. Successful selection was analyzed by phage pool ELISA in medium-bound microtiter plates coated with ferritin (125 ng / well), hIgG1-Fc (125 ng / well), BSA (125 ng / well), or Sigmablocker (Greiner Bio-One). Bound phages were detected using α-M13HRP-conjugated antibody (GE Healthcare).

[0096] Cloning of target-binding phage pools into expression vectors. A selection pool that specifically binds to ferritin in a phage pool ELISA is amplified by PCR according to methods known in the art, cleaved with an appropriate restriction nuclease, and conjugated to a derivative of the expression vector pET-28a (Merck, Germany) containing an N-terminal GFP-10xHis tag, followed by a TVMV cleavage site and a C-terminal cysteine.

[0097] Results: Various phage display selection pools produced specific signals for their respective ON target ferritin. Controls containing BSA or Sigmablocker did not show binding for almost all pools. The selected pools were sequenced and subcloned for high-throughput screening.

[0098] Primary screening: Eight selection pools were advanced to high-throughput screening for ferritin. Therefore, ferritin [c=3.0 μg / ml] was immobilized in 384-well high-binding plates, and bound mutants were detected by fluorescence signaling (excitation 485 nm / emission 535 nm). 2141 mutants were selected for secondary screening of ferritin (ON target) and BSA (OFF target), and the binding of these mutants was detected by fluorescence signaling (excitation 485 nm / emission 535 nm). Specific mutants were selected for sequencing, μ-scale purification, and BLI analysis. Hit criteria: Soluble protein expression, sample signal greater than 5.000, and 5 times greater than the OFF target signal.

[0099] μ-scale purification and BLI analysis: 376 mutants resulting from secondary screening were defined as hits for ferritin obtained through μ-scale purification and BLI analysis using an Octet 8 channel system. For BLI measurements, mutants were immobilized on the surface of a Ni-NTA sensor (ForteBio) using His-Tag. Upon binding, the mutants accumulated on the surface, increasing the refractive index. This change in refractive index was measured in real time and plotted as an nm shift over time. The affinity of the captured mutants for ferritin [c=200nM] was determined and also plotted as an nm shift over time. These proteins were selected for subcloning in a format suitable for affinity chromatography and mass production (fusion proteins).

[0100] Example 2. Expression and purification of a fusion protein containing ferritin-binding protein. A fusion protein containing ferritin-binding protein was expressed in the Escherichia coli (BL21) (DE3) fermentation process using the pNP-016 vector system under the control of the T7 promoter. Seed cultures were grown in a preliminary culture medium (34.5 g / L yeast extract, 0.61 g / L MgSO4, 14.2K2HPO4, 0.5 g / L NH4Cl, 50 μg / mL kanamycin). The fermentation process was carried out in a parallel benchtop bioreactor, such as a fed-batch culture. Seed cultures were seeded into a medium (17.25 g / L yeast extract, 0.61 g / L MgSO4, 14.2K2HPO4, 0.5 g / L NH4Cl, 50 μg / mL kanamycin), and the cultures were grown until the culture medium was depleted (37°C, pH 7.1, 30% pO2 saturated, aerated 2VVM). Logarithmic growth was performed using glucose as the primary culture medium (200 g / L glucose, 276 g / L yeast extract, 1.1 g / L MgSO4, 50 μg / mL kanamycin). Protein expression was induced at a constant supply rate and 30°C for 5 hours using isopropyl β-D-1-thiogalactopyranoside (IPTG, final concentration 1 mM). To recover biomass, cells were centrifuged at 12,000 x g for 30 minutes. Bacterial pellets were stored at -20°C before processing. Expression was analyzed by SDS-PAGE.

[0101] Fusion proteins 219124 and 219125 were purified by cation exchange chromatography and molecular sieving. After cell disruption, the pH was adjusted to 3.0 and 2.5, respectively, to precipitate most of the host protein. The initial capture step was performed at pH 3.5 using SP Sepharose HP (Cytiva; binding buffer: 20 mM citrate, 1 mM EDTA pH 3.5, elution buffer: 20 mM citrate, 1 mM EDTA, 1 M NaCl pH 3.5), followed by molecular sieving chromatography (Superdex 75 26 / 600, Cytiva) in 20 mM citrate, 150 mM NaCl, 1 mM EDTA pH 6.0 performed on an AKTA avant system (Cytiva).

[0102] Fusion proteins 219126 and 219127 were purified by gel filtration. After cell disruption, acetic acid was added until the final concentration reached 100 mM, and then the pH was adjusted to 2.5 and 2.0, respectively. The final finishing step was performed using molecular sieve chromatography (Sephacryl 200HR XK26 / 70, Cytiva) in 20 mM citrate, 150 mM NaCl, and 1 mM EDTA at pH 6.0 using an AKTA avant system (Cytiva).

[0103] After SDS-PAGE analysis, the positive fraction was pooled, and the protein concentration was determined by absorbance measurement at 280 nm using the molar absorption coefficient. Further analysis included RP-HPLC and SE-HPLC. Reverse-phase chromatography (RP-HPLC) was performed using an Ultimate 3000 HPLC system (Thermo Fisher Scientific) and a PLRP-S (5 μm, 300 Å) column (Agilent). The purity of all obtained proteins was 100%. Analytical molecular sieve chromatography (SE-HPLC) was performed using an Ultimate 3000 HPLC system (Thermo Fisher Scientific) and a Superdex75 increase 5 / 150 GL (Cytiva). No aggregates or oligomers were obtained. The final protein yield was 0.95–4.14 mg / g wet biomass.

[0104] Example 3. Binding analysis of fusion proteins by SPR. After NHS / EDC activation using the Biacore 3000 SPR or Sierra SPR-32 system (Bruker), the purified fusion protein was immobilized onto a CM5 (Cytiva) or High Capacity Amine sensor chip (Bruker) using PDEA. The chip was equilibrated with SPR running buffer (PBS 0.05% Tween pH 7.3 or 10 mM sodium acetate, 150 mM NaCl, 0.05% Tween, pH 4.5). Upon binding, the target analyte accumulated on the surface, increasing the refractive index. This change in refractive index was measured in real time and plotted as a response to time or as a resonance unit. The analyte, ferritin protein, was applied to the chip at a flow rate of 30 μl / min in serial dilutions. Association was performed for 120 seconds, followed by dissociation for 120 seconds. After each run, the surface of the chip was regenerated with 30 μl of regeneration buffer (6 M guanidine hydrochloride pH 4.0) and equilibrated with running buffer. The coupling tests were performed using Biacore 3000 Control Software or the Sierra SPR-32 system (Bruker), and the data evaluation was performed using the Langmuir 1:1 model (RI=0) with BIAevaluation or Sierra Analyser software provided by the manufacturer. The evaluated dissociation constant (K) D The values ​​were standardized for the immobilized protein and shown. The change in refractive index measured in real time is shown and plotted as the response to time [seconds] or resonance unit [RU]. The fusion protein was ferritin with K1 nM at pH 4.5 and pH 7.3. D The proteins bound to ferritin (see Table 1). Figure 2 shows that fusion protein 219127 bound to ferritin with high affinity at pH 4.5.

[0105] TIFF0007836119000001.tif60170

[0106] Example 4. Affinity Purification of Ferritin Binding parameters. Fusion proteins 219124, 219126, and 219127 were uniformly purified and immobilized on 20 mg / mL activated Praesto® Epoxy 85 (Purolite) according to the manufacturer's instructions. Binding conditions: 35°C for 3 hours, pH 9.5, per mL of resin. All ligands bound well to epoxy-activated Praesto 85 resin. Binding densities ranged from 9.9 to 16.3 mg / ml for each bound ligand.

[0107] Ferritin affinity chromatography. For affinity chromatography, Praesto 85-219127 was packed into a superformance column housing (Gotec, 5-50) and equilibrated with 1x PBS pH 7.3. 30 mL of a solution containing ferritin (e.g., at least 500 μg of human ferritin, Merck, catalog no.: 341482) was loaded with a residence time of 6 minutes. A washing step with 100 mM sodium acetate pH 4.5 was included to confirm that ferritin binds to the ligand even at pH 4.5. Elution was performed using 100 mM citrate on a gradient from pH 4.5 to pH 2.0 over 15 column volumes (CV). The pH of the buffer fraction containing the target was determined.

[0108] Fusion protein 219127 (including SEQ ID NOs. 6 and 2) showed a uniform single-peak elution profile (see Figure 3A).

[0109] The maximum peak in the pH gradient profile was at pH 3.1. Ferritin eluted by SDS-PAGE showed high purity (Figure 3B). Highly concentrated ferritin was observed compared to the initial load. The identity of ferritin in the eluted fraction was confirmed by specific ferritin ELISA (IBL international; catalog number: DB59111, according to the manufacturer's instructions).

[0110] Static binding capacity (SBC). Resins containing immobilized fusion proteins 219124, 219126, or 219127 (Praesto85_219214, Praesto85_219126, Praesto85_219127) were equilibrated in 1x PBS pH 7.3. Praesto85_219214, Praesto85_219126, or Praesto85_219127 purified human ferritin (Merck, catalog number: 341482, 1.5 mg / ml, 400 μl, PBS pH 7.3) was mixed with 20 μl of resin at room temperature for 1 hour. The matrix was washed twice with 300 μl of 1x PBS pH 7.3, and the bound proteins were eluted with 3 x 100 μl of 100 mM citrate pH 2.0. The amount of eluted ferritin was calculated using ferritin ELISA (IBL International, catalog number DB59111, according to the manufacturer's instructions).

[0111] SBC was determined by the calculated amount of eluted protein. The static binding capacity was 5.6 mg / ml for both Praesto85_219126 and Praesto85_219127, and 9.6 mg / ml for Praesto85_219124.

[0112] array Sequence ID 1: Ferritin-binding protein AKFDXXQXYADXXILXLXNLTEEQRNAFRQSLSDDPSVSWXVLXEARXLNEXQAPK X may be any natural amino acid. As used herein, X may be any one of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V. Sequence ID 2: Ferritin-binding protein (218990) AKFDQWQSYADHQILKLPNLTEEQRNAFRQSLSDDPSVSWEVLWEARELNESQAPK Sequence ID 3: Ferritin-binding protein (218989) AKFDAYQSYADKEILQLPNLTEEQRNAFRQSLSDDPSVSWEVLQEARVLNESQAPK Sequence ID No. 4: Ferritin-binding protein (218894) AKFDQFQLYADKEILELFNLTEEQRNAFRQSLSDDPSVSWYVLWEARMLNEAQAPK Sequence ID 5: Fusion protein containing Sequence ID 2 (219126) AKFDQWQSYADHQILKLPNLTEEQRNAFRQSLSDDPSVSWEVLWEARELNESQAPKIAAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKIAAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKC Sequence ID 6: Fusion protein containing Sequence ID 2 (dimer) (219127) AKFDQWQSYADHQILKLPNLTEEQRNAFRQSLSDDPSVSWEVLWEARELNESQAPKIAAKFDQWQSYADHQILKLPNLTEEQRNAFRQSLSDDPSVSWEVLWEARELNESQAPKI AAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKIAAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKC SEQ ID NO: 7: Fusion protein containing SEQ ID NO: 3 (219124) AKFDAYQSYADKEILQLPNLTEEQRNAFRQSLSDDPSVSWEVLQEARVLNESQAPKIAAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKIAAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKC Sequence ID 8: Fusion protein containing Sequence ID 3 (dimer) (219125) AKFDAYQSYADKEILQLPNLTEEQRNAFRQSLSDDPSVSWEVLQEARVLNESQAPKIAAKFDAYQSYADKEILQLPNLTEEQRNAFRQSLSDDPSVSWEVLQEARVLNESQAPKI AAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKIAAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKC Sequence ID 9: Fusion protein containing Sequence ID 4 (219122) AKFDQFQLYADKEILELFNLTEEQRNAFRQSLSDDPSVSWYVLWEARMLNEAQAPKIAAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKIAAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKC SEQ ID NO: 10: Fusion protein containing SEQ ID NO: 4 (dimer) (219123) AKFDQFQLYADKEILELFNLTEEQRNAFRQSLSDDPSVSWYVLWEARMLNEAQAPKIAAKFDQFQLYADKEILELFNLTEEQRNAFRQSLSDDPSVSWYVLWEARMLNEAQAPKI AAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKIAAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKC Sequence ID 11: Non-ferritin-binding protein AQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPK Sequence ID 12: Ferritin MTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLEFPSPISPSPSCWHHYTTNRPQPQHHLLRPRRRKRPHSIPTPILIFRSP

Claims

1. A ferritin-binding protein comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 3, wherein the binding protein has a binding affinity of less than 100 nM to ferritin.

2. The binding protein according to claim 1, wherein two, three, four, five, or six binding proteins are linked to one another.

3. The binding protein according to any one of claims 1 to 2, wherein the binding protein is fused to at least one non-ferritin binding protein.

4. A fusion protein comprising at least one binding protein and at least one non-ferritin binding protein as described in claim 1.

5. The fusion protein according to claim 4, wherein the fusion protein includes an attachment site for site-specific binding to a solid support.

6. A ferritin-binding protein according to any one of claims 1 to 2 or a fusion protein according to any one of claims 4 to 5 for use in technologies such as affinity chromatography.

7. An affinity separation matrix comprising a ferritin-binding protein according to any one of claims 1 to 2 or a fusion protein according to any one of claims 4 to 5.

8. Use of a ferritin-binding protein according to any one of claims 1 to 2 or a fusion protein according to any one of claims 4 to 5 for affinity chromatography.

9. A method for purifying ferritin, wherein the method is: (i) To provide a liquid containing ferritin, (ii) To provide an affinity separation matrix comprising at least one ferritin-binding protein according to any one of claims 1 to 2 or a fusion protein according to any one of claims 4 to 5, bound to the affinity separation matrix. (iii) Contacting the affinity separation matrix with the liquid under conditions that enable the binding of at least one ferritin-binding protein according to any one of claims 1 to 2 or the fusion protein according to any one of claims 4 to 5, and (iv) A method comprising eluting the ferritin from the affinity separation matrix.

10. A method for analyzing the presence or absence of ferritin, comprising the use of a ferritin-binding protein according to any one of claims 1 to 2 or a fusion protein according to any one of claims 4 to 5.

11. A method for analyzing the presence or absence of ferritin in a liquid sample, the method comprising the following steps: (i) A step of providing a liquid containing ferritin, (ii) A step of providing a ferritin-binding protein according to any one of claims 1 to 2 or a fusion protein according to any one of claims 4 to 5. (iii) The step of bringing the liquid of (i) into contact with the ferritin-binding protein according to any one of claims 1 to 2 or the fusion protein according to any one of claims 4 to 5 under conditions that enable the ferritin-binding protein to bind to the ferritin, (iv) the step of isolating the ferritin and binding protein complex, and (v) The step of determining the amount of the ferritin-binding protein in the liquid of (i), method.

12. A nucleic acid molecule encoding a ferritin-binding protein according to any one of claims 1 to 2 or a fusion protein according to any one of claims 4 to 5.

13. A vector comprising the nucleic acid molecule described in claim 12.

14. A host cell or non-human host comprising the ferritin-binding protein according to any one of claims 1 to 2 or the fusion protein according to any one of claims 4 to 5.

15. A method for producing a ferritin-binding protein according to any one of claims 1 to 2 or a fusion protein according to any one of claims 4 to 5, comprising culturing a host cell according to claim 14 under appropriate conditions to obtain a ferritin-binding protein or a fusion protein containing the ferritin-binding protein, and optionally isolating the ferritin-binding protein or a fusion protein containing the ferritin-binding protein.

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