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 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. [Background technology]

[0002] Ferritin is a large, multi-subunit protein complex and 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 serious effects on, for example, neurological development in children.

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

[0004] Purified ferritin can be administered to individuals in need of iron and used to treat or supplement iron deficiency disorders.

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

[0006] Thus, there is a strong need in the art to provide an efficient method for the purification of ferritin. The present invention meets this need by providing a novel ferritin-binding protein that is particularly advantageous since it allows for the precise purification of ferritin, in particular by affinity chromatography, for further use in medical applications.

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

[0008] The present disclosure provides the following items 1 to 15, but is not limited thereto: 1. A ferritin binding protein comprising an amino acid sequence having at least 90% sequence identity to any one selected from the group consisting 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 for ferritin of less than 100 nM. 2. The binding protein according to item 1, wherein 2, 3, 4, 5 or 6 binding proteins are linked to each other. 3. The binding protein according to items 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 according to claim 1 and at least one non-ferritin binding protein. 5. The fusion protein according to item 4, wherein the fusion protein comprises an attachment site for site-specific binding to a solid support. 6. A ferritin-binding protein according to any one of items 1 to 3 or a fusion protein according to items 4 to 5 for use in technical applications such as affinity chromatography. 7. An affinity separation matrix comprising the ferritin-binding protein according to any one of items 1 to 3 or the fusion protein according to items 4 to 5. 8. Use of the ferritin-binding protein according to any one of items 1 to 3 or the fusion protein according to items 4 to 5 or the affinity separation matrix according to item 8 for affinity chromatography. 9. A method for purifying ferritin, comprising: (i) providing a liquid containing ferritin; (ii) providing an affinity separation matrix comprising at least one ferritin-binding protein according to any one of items 1 to 3 or a fusion protein according to items 4 to 5 bound to said affinity separation matrix, (iii) contacting the affinity separation matrix with the liquid under conditions that allow binding of at least one ferritin-binding protein according to any one of items 1 to 3 or a fusion protein according to items 4 to 5; and (iv) eluting the ferritin from the affinity purification matrix. 10. Use of a ferritin-binding protein according to items 1 to 3 or a fusion protein according to items 4 to 5 or an affinity matrix according to 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 steps of: (i) providing a liquid containing ferritin; (ii) providing a ferritin-binding protein according to items 1 to 3 or a fusion protein according to items 4 to 5; (iii) contacting the liquid of (i) with a ferritin-binding protein according to items 1 to 3 or a fusion protein according to items 4 to 5 under conditions allowing binding of the ferritin-binding protein; (iv) isolating the complex of ferritin and the binding protein; and (v) determining the amount of ferritin-binding protein in the liquid of (i); method. 12. A nucleic acid molecule encoding the ferritin-binding protein according to items 1 to 3 or the fusion protein according to items 4 to 5. 13. A vector comprising the nucleic acid molecule according to item 12. 14. A host cell or a non-human host comprising the ferritin-binding protein according to items 1 to 3 or the fusion protein according to items 4 to 5, the nucleic acid according to item 12, and / or the vector according to item 13. 15. A method for producing a ferritin-binding protein according to items 1 to 3 or a fusion protein according to items 4 to 5, comprising culturing a host cell according to item 14 under suitable conditions to obtain said ferritin-binding protein or a fusion protein comprising said ferritin-binding protein, and optionally isolating said ferritin-binding protein or a fusion protein comprising said ferritin-binding protein.

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

[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 multiple sequence alignment tool. [Diagram 2] Figure 2 shows SPR of 219127 (a fusion protein containing SEQ ID NO:2) immobilized via the c-terminal cysteine ​​residue using PDEA conjugation. 219127 binds ferritin with an affinity of 0.5 nM or less even at pH 4.5. [Diagram 3]Figure 3 shows the purification of ferritin by 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 shows the UV 280 nm signal. The gradient of the elution buffer is shown with a dashed black line. The figure shows a homogenous elution profile of ferritin with a single peak. The maximum peak of the pH gradient profile was at pH 3.1. Figure 3B shows an SDS-PAGE of the analyzed fractions of an affinity chromatography run using Praesto85-219127. The liquid containing ferritin was loaded. The purified human ferritin is visible as a MW band of 20 kDa (arrow). No significant impurities could be detected. Lane 1: protein marker, lane 2: ferritin, lanes 3-7: flow-through, lane 8: wash fraction (washed at pH 4.5), lanes 9-10 elution fraction. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention provides a novel protein that has specific binding affinity to ferritin.The novel protein of the present invention is particularly advantageous because, as an affinity ligand for ferritin, it allows for precise purification of ferritin, for example, in affinity chromatography.Any polypeptide selected from the group of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, or an amino acid sequence having at least 90% identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively, binds with high affinity to human ferritin.

[0012] Before the present invention is described in more detail below, it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, as they may vary. It should also be understood that the terms used herein are only for the purpose of describing specific aspects and embodiments, and are not intended to limit the scope of the present invention, as set forth by the accompanying documents. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. This includes those working in the field of protein engineering and purification, but also those working in the field of developing new specific ferritin-binding molecules for use in technical applications, for example, for use as affinity ligands in affinity chromatography.

[0013] Preferably, the terms used herein are defined as 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 this specification and subsequent documents, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" are understood to denote the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps. The terms "comprise(s)" or "comprising" may include the limitation to "consists of" or "consisting of" if such a limitation is necessary for any reason or to any extent.

[0015] Throughout the specification of the present invention, several documents (e.g., patents, patent applications, scientific literature, manufacturer's specifications, instructions, UniProt Accession Numbers, etc.) may be cited. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by prior invention. Some of the documents cited in the present invention may be characterized as "incorporated by reference." In the event of a conflict between the definitions or teachings of such incorporated references and the definitions or teachings recited in this specification, the language of this specification shall control.

[0016] All sequences referenced herein are disclosed in the accompanying sequence listing, which by its content and disclosure in its entirety forms part of the disclosure of this specification.

[0017] General definitions of important terms used herein The term "ferritin" refers to the amino acid sequence set forth in UniProtKB Q8TD27. The term "ferritin" includes all polypeptides exhibiting 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 that has a detectable interaction with ferritin, as determined by a suitable method, such as, for example, SPR analysis or BLI, or other suitable technique known to one of skill 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 a suitable method, such as, for example, SPR analysis or BLI or other suitable technique known to one of skill in the art.

[0020] The term "binding affinity" refers to the ability of a polypeptide of the present invention to bind to ferritin. Binding affinity is typically measured using the equilibrium dissociation constant (K D ) and reported. Binding affinity and dissociation constants can be quantitatively measured. Methods for measuring binding affinity are well known to those skilled in the art and can be selected, for example, from the following established methods in the art: surface plasmon resonance (SPR), biolayer interferometry (BLI), enzyme-linked immunosorbent assay (ELISA), equilibrium exclusion analysis (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 is measured at temperatures ranging from 20°C to 30°C. Unless otherwise specified, the K D is determined by SPR at 25° C. In various embodiments of the invention, binding affinity to ferritin may be determined by a Sierra SPR-32 system (Bruker).

[0021] The term "fusion protein" refers to a protein comprising at least a first protein genetically linked to at least a second protein. A fusion protein is created by joining two or more genes that originally encoded separate proteins. Thus, a fusion protein may comprise multimers of the same or different proteins expressed as a single linear polypeptide.

[0022] The term "amino acid sequence identity" refers to a quantitative comparison of the identity (or difference) of the amino acid sequences of two or more proteins. "Percentage of amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity. To determine sequence identity, the sequence of the query protein is aligned to the sequence of the reference protein or polypeptide, e.g., the polypeptide 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, e.g., to the amino acid sequence of SEQ ID NO:1, it is preferable to utilize the freely available SIM local similarity program (Xiaoquin Huang and Webb Miller (1991), Advances in Applied Mathematics, vol. 12: 337-357). For multiple sequence alignments, it is preferred to use ClustalW (Thompson et al. (1994) Nucleic Acids Res., 22(22):4673-4680).

[0023] The terms "protein" and "polypeptide" refer to any chain of two or more amino acids linked by peptide bonds and do not refer to a specific length of the product. Thus, "peptide," "protein," "amino acid chain," or any other term used to refer to a chain of two or more amino acids, are 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, for example, 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 check this by incubating the ferritin-binding protein with, for example, sodium hydroxide solution as described in the examples, and then testing the binding affinity to ferritin by routine experiments known to those skilled in the art, such as, for example, chromatographic methods.

[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 and moves them across or through a stationary phase (e.g., a solid matrix). The molecules in the mobile phase can be separated due to differences in the interactions of different types of molecules in the mobile phase with the stationary phase.

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

[0027] The terms "affinity matrix" or "affinity purification matrix", as used interchangeably herein, refer to a matrix, such as, for example, a chromatography matrix, onto which an affinity ligand (e.g., a ferritin-binding protein or a fusion protein containing a ferritin-binding protein) is attached. The attached affinity ligand is capable of specifically binding to a molecule of interest (e.g., ferritin) to be purified or removed from the liquid. The liquid may be, for example, 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, which removes it from the liquid.

[0029] DETAILED DESCRIPTION OF EMBODIMENTS OF THE PRESENTINVENTION The present invention is further described. In the following passages, various aspects of the present invention are defined in more detail. Each aspect defined below may be combined with any other aspect or aspects, unless expressly stated otherwise. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0030] The novel ferritin-binding protein exhibits specific binding affinity to ferritin. The ferritin-binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, or an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

[0031] In some embodiments, the ferritin binding protein comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the ferritin binding protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1.

[0032] In some embodiments, the ferritin binding protein comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the ferritin binding protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2.

[0033] In some embodiments, the ferritin binding protein comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the ferritin binding protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3.

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

[0035] In some embodiments, the ferritin binding protein comprises at least one amino acid sequence shown in FIG.

[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 to any one of the amino acid sequences of SEQ ID NOs:1-4.

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

[0038] The amino acid corresponding to position 5 of SEQ ID NO:1 may be selected from Q or A.

[0039] The amino acid corresponding to position 6 of SEQ ID NO: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, preferably S.

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

[0042] The amino acid corresponding to position 13 of SEQ ID NO: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 SEQ ID NO:1 may be selected from E or Y, preferably E.

[0045] The amino acid corresponding to position 44 of 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 to any one selected from the group of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO: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 NO:2 and SEQ ID NO:3.

[0049] One embodiment refers to a ferritin binding protein comprising an amino acid sequence having at least 90% sequence identity to 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 to 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 unaltered.

[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 to 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, 48 of SEQ ID NO:2 or SEQ ID NO:3 are modified.

[0052] One advantage of the ferritin-binding protein disclosed herein is the important functional property that the protein specifically binds to ferritin with low affinity.Not to mention, this is particularly advantageous when purifying ferritin in a protein purification process.The ferritin-binding protein is functionally characterized by a binding affinity to ferritin of less than 100nM.In some embodiments, the ferritin-binding protein is functionally characterized by a binding affinity to ferritin of less than 50nM.In some embodiments, the ferritin-binding protein is functionally characterized by a binding affinity to ferritin of less than 10nM.

[0053] Multimers. In one embodiment of the present invention, the ferritin-binding protein comprises 1, 2, 3, 4, 5, or 6 binding proteins linked together. In one embodiment of the present invention, the ferritin-binding protein comprises one ferritin-binding protein or two ferritin-binding proteins linked together. The binding protein multimers are artificially created by recombinant DNA techniques, which are generally well known to those skilled in the art. In some embodiments, the multimers are homomultimers, e.g., the amino acid sequences of the ferritin-binding proteins are identical. In other embodiments, the multimers are heteromultimers, e.g., the amino acid sequences of the ferritin-binding proteins are different.

[0054] Fusion protein. In some embodiments, the above-mentioned ferritin binding protein is fused with one additional polypeptide different from the disclosed polypeptide. In various embodiments, the additional polypeptide different from the ferritin binding protein disclosed herein may be a non-ferritin binding protein. In some embodiments, the additional non-ferritin binding polypeptide is a non-immunoglobulin (Ig) binding protein, such as, but not limited to, a protein that does not bind Ig. In some embodiments, a common structural feature of the non-ferritin binding protein is that it has a triple helix protein A-like structure that is 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 to SEQ ID NO: 11. In some embodiments, the non-ferritin binding protein has at least 89% amino acid sequence identity to SEQ ID NO: 11.

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

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

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

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

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

[0061] In one embodiment, the fusion protein comprises 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 comprises 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 comprises 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 an attachment site 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 comprise 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. Exemplary of such sequences include, but are not limited to, Strep-tag, oligohistidine-tag, glutathione S-transferase, maltose-binding protein, intein, intein fragment, or albumin-binding domain of protein G, or others. In one embodiment, the additional amino acid sequence comprises one or more peptide sequences that confer affinity to a particular chromatographic column material. The ferritin-binding protein or fusion protein comprising the ferritin-binding protein may comprise a specific attachment site, preferably at the C-terminus, for attachment to a solid support, such as cysteine ​​or lysine.

[0069] Uses of Novel Ferritin Binding Proteins in Technical Applications Also provided herein are uses of any of the 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 called affinity purification) utilizes specific binding interactions between molecules. Protein immobilization and affinity chromatography methods 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 an affinity separation matrix comprising at least one ferritin-binding protein as described above or a fusion protein as described above bound to the affinity separation matrix; (iii) contacting the affinity separation matrix with a liquid under conditions allowing binding of the at least one ferritin-binding protein as described above or the fusion protein as 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, the liquid being plasma or serum or being obtained from plasma or serum; (ii) providing an affinity separation matrix comprising at least one ferritin-binding protein as described above or a fusion protein as described above bound to the affinity separation matrix; (iii) contacting the affinity separation matrix with the liquid under conditions that allow binding of the at least one ferritin-binding protein as described above or the fusion protein as described above; and (iv) eluting the ferritin from the affinity purification matrix.

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

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

[0075] Attachment to a solid support. In various aspects and / or embodiments of the invention, the novel ferritin-binding proteins disclosed herein, including novel ferritin-binding proteins made or obtained by any of the methods described above, are attached to a solid support. In some embodiments, the ferritin-binding protein comprises an attachment site for site-specific covalent attachment of the ferritin-binding protein to a solid support. Specific attachment sites include, but are not limited to, natural amino acids such as cysteine ​​or lysine, which allow for specific chemical reactions with reactive groups on 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 comprises a ferritin binding protein or a fusion protein comprising a ferritin binding protein.

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

[0078] Solid support matrices for affinity chromatography are well known in the art and include, but are not limited to, agarose and stabilized derivatives of agarose, 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 of the above.

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

[0080] In one embodiment, the matrix is ​​composed of substantially spherical beads, such as sepharose or agarose beads. The particulate form of the matrix can be used as a packed bed or in a floating form, including an expanded bed. In other embodiments of the invention, the solid support matrix is ​​a membrane, such as a hydrogel membrane. In some embodiments, affinity purification can include a membrane as a matrix to which the ferritin binding protein described herein is covalently attached. The solid support can also be in the form of a membrane in a cartridge.

[0081] In some embodiments, affinity purification involves a chromatography column containing a solid support matrix to which the novel protein of the present invention is covalently bound. The above-mentioned ferritin-binding protein or a fusion protein containing a ferritin-binding protein may be attached to a suitable solid support matrix by conventional binding techniques. Methods for immobilizing protein ligands to solid supports are well known in the field 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, reference is made to the use of the above-mentioned ferritin-binding protein or the above-mentioned fusion protein or the above-mentioned affinity matrix in a method for determining the presence or absence of ferritin.

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

[0084] A method for quantifying ferritin. A further embodiment relates to a method for quantifying ferritin, which comprises: (i) providing a liquid containing ferritin; (ii) providing a matrix to which a ferritin-binding protein or a fusion protein comprising a ferritin-binding protein as described herein is covalently attached; (iii) contacting the affinity purification matrix with the liquid under conditions that allow the binding of at least one ferritin-binding protein or a fusion protein comprising 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), enzyme reaction, surface plasmon resonance (SPR), or chromatography, but are not limited thereto.

[0085] Polynucleotides, Vectors, Host Cells. One embodiment includes an isolated polynucleotide or nucleic acid molecule that encodes a ferritin-binding protein or a fusion protein that includes a ferritin-binding protein as described herein. Further embodiments also include proteins encoded by the polynucleotides.

[0086] Further provided are vectors, particularly expression vectors, comprising the isolated polynucleotides or nucleic acid molecules of the ferritin-binding proteins or fusion proteins comprising ferritin-binding proteins described herein, as well as host cells comprising the isolated polynucleotides or expression vectors. For example, one or more polynucleotides encoding the ferritin-binding proteins or fusion proteins comprising ferritin-binding proteins disclosed herein may be expressed in a suitable host, and the produced proteins may be isolated. By vector, we mean any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) that can be used to transfer protein-encoding information to a host cell. Suitable vectors that may be applied are known in the art.

[0087] Suitable host cells include prokaryotic or eukaryotic organisms, such as bacterial host cells carrying vectors, yeast host cells or non-human host cells. A suitable host may be a microbial expression host capable of expressing the protein of the invention. Suitable bacterial expression host cells or systems are known in the art. Various mammalian or insect cell culture systems known in the art may also be utilized to express recombinant proteins.

[0088] Further provided is an isolated cell comprising a polynucleotide or nucleic acid, or vector disclosed herein.

[0089] Methods for producing the proteins of the invention. In a further embodiment, a method for producing the described ferritin-binding protein or a fusion protein comprising a ferritin-binding protein is provided, the method comprising (i) culturing a (suitable) host cell under suitable conditions for expressing the ferritin-binding protein or the fusion protein comprising a ferritin-binding protein to obtain the ferritin-binding protein or the fusion protein comprising a ferritin-binding protein, and (ii) optionally isolating the ferritin-binding protein or the fusion protein comprising a ferritin-binding protein. Suitable 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 technique, such as simple organic synthesis methods, solid-phase assisted synthesis techniques, or by commercially available automated synthesizers. These proteins may also be prepared using conventional recombinant techniques, either alone or in combination with conventional synthesis techniques.

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

[0092] The following examples are provided to further illustrate the present invention, but the present invention is not limited thereto, and the following examples are merely intended to demonstrate the feasibility of the present invention based on the above description. EXAMPLES

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

[0094] Example 1. Selection and Screening Libraries. Dedicated cDNA libraries based on stable protein A-like variants (artificial mosaic proteins composed of fragments of the protein A domain and additional mutations) were synthesized in-house with randomized oligonucleotides by synthetic trinucleotide phosphoramidites (ELLA Biotech) or synthesized externally by Geneart to simultaneously exclude cysteine ​​and other amino acid residues at randomized positions to achieve a balanced amino acid distribution. For the following selection process by phage display, the corresponding cDNA libraries were amplified by PCR and ligated into pCD33-OmpA phagemid. An aliquot of the ligation mixture was used for electroporation of E. coli SS320 to generate and purify the phage libraries, which were then stored as cryopreservations.

[0095] Selection by phage display. Established recombinant genetic methods were used unless otherwise stated. Naive libraries were enriched for human ferritin (Calbiochem instructions, CAS 9007-73-2) as ON target using phage display as selection system. At each round, a pre-selection step was performed using empty magnetic Dynabeads™ M-270 Epoxy blocked with Sigmablocker. The AIT method was applied, which means 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 low-temperature 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 on an automated KingFisher system (Thermo Fisher) to isolate and capture the desired phage-target complexes. Target concentrations started at 40 nM (round 1) and were decreased to 10 nM in each round (round 3). Bound phages were eluted with trypsin and re-amplified. Selection success was analyzed by phage pool ELISA in medium-binding microtiter plates (Greiner Bio-One) coated with ferritin (125 ng / well), hIgG1-Fc (125 ng / well), BSA (125 ng / well) or Sigmablocker. Bound phages were detected using an α-M13 HRP-conjugated antibody (GE Healthcare).

[0096] Cloning of target-binding phage pools into expression vectors. Selected pools showing specific binding to ferritin in phage pool ELISA are amplified by PCR according to methods known in the art, cleaved with appropriate restriction nucleases, and ligated into 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: The various phage display selected pools gave specific signals for their respective ON target ferritin. Controls containing BSA or Sigmablocker showed no binding for almost all pools. Selected pools were sequenced and subcloned for high-throughput screening.

[0098] Primary screening: Eight selected pools were advanced to high-throughput screening against ferritin. Thus, ferritin [c=3.0 μg / ml] was immobilized on a 384-well high-binding plate and bound variants were detected by fluorescent signal (excitation 485 nm / emission 535 nm). 2141 variants were selected for secondary screening against ferritin (ON target) and BSA (OFF target) where binding of variants was detected by fluorescent signal (excitation 485 nm / emission 535 nm). Specific variants were selected for sequencing, μ-scale purification and BLI analysis. Hit criteria: soluble protein expression, sample signal >5.000 and 5-fold greater than the signal of the OFF target.

[0099] μ-scale purification and BLI analysis: 376 mutants resulting from the secondary screen were defined as hits for μ-scale purification and BLI analysis against ferritin using the Octet8 channel system. For BLI measurements, mutants were immobilized on the surface of a Ni-NTA sensor (ForteBio) by His-Tag. Upon binding, mutants accumulated on the surface, increasing the refractive index. This change in refractive index was measured in real time and plotted as nm shift versus time. The affinity of the captured mutants for ferritin [c=200 nM] was determined and also plotted as nm shift versus time. These proteins were selected for affinity chromatography applications and subcloning in a suitable format (fusion protein) for large-scale production.

[0100] Example 2. Expression and purification of fusion proteins containing ferritin-binding proteins A fusion protein containing a ferritin-binding protein was expressed in an Escherichia coli BL21 (DE3) fermentation process using the pNP-016 vector system under the control of the T7 promoter. The seed culture was grown in a preculture medium (34.5 g / L yeast extract, 0.61 g / L MgSO4, 14.2 K2HPO4, 0.5 g / L NH4Cl, 50 μg / mL kanamycin). The fermentation process was carried out in parallel bench-top bioreactors as a fed-batch process. The medium (17.25 g / L yeast extract, 0.61 g / L MgSO4, 14.2 K2HPO4, 0.5 g / L NH4Cl, 50 μg / mL kanamycin) was inoculated with the seed culture and the culture was grown (37 °C, pH 7.1, 30% pO2 saturation, 2 VVM with aeration) until the medium was exhausted. Exponential growth was performed in a feeding regime with glucose as the main culture medium (200 g / L glucose, 276 g / L yeast extract, 1.1 g / L MgSO4, 50 μg / mL kanamycin). Protein expression was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG, final concentration 1 mM) at a constant feed rate for 5 h at 30 °C. To harvest biomass, cells were centrifuged at 12,000 x g for 30 min. 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. An 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 run on an AKTA avant system (Cytiva).

[0102] Fusion proteins 219126 and 219127 were purified by gel filtration. After cell disruption, acetic acid was added to a final concentration of 100 mM, followed by pH adjustment to pH 2.5 and 2.0, respectively. The final polishing step was performed by size exclusion chromatography (Sephacryl 200HR XK26 / 70, Cytiva) in 20 mM citric acid, 150 mM NaCl, 1 mM EDTA pH 6.0 using an AKTA avant system (Cytiva).

[0103] After SDS-PAGE analysis, positive fractions were pooled and protein concentrations were determined by absorbance measurements at 280 nm using the molar absorption coefficient. Further analyses 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 proteins obtained 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 yields were 0.95-4.14 mg / g wet biomass.

[0104] Example 3. Binding analysis of fusion proteins by SPR After NHS / EDC activation with a Biacore 3000 SPR or Sierra SPR-32 system (Bruker), purified fusion proteins were immobilized on a sensor chip CM5 (Cytiva) or a 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 response or resonance units versus time. Analyte ferritin protein was applied to the chip in serial dilutions at a flow rate of 30 μl / min. Association was performed for 120 s and dissociation was performed for 120 s. After each run, the surface of the chip was regenerated with 30 μl regeneration buffer (6 M guanidine-HCl pH 4.0) and equilibrated with running buffer. Binding studies were performed by using Biacore 3000 Control Software or Sierra SPR-32 system (Bruker) and data evaluation was performed by BIAevaluation or Sierra Analyser software provided by the manufacturer using the Langmuir 1:1 model (RI=0). The estimated dissociation constants (K D ) was normalized to the immobilized protein and shown. The change in refractive index measured in real time is shown and plotted as response or resonance units [RU] versus time [sec]. The fusion protein bound to ferritin with a K of less than 1 nM at pH 4.5 and pH 7.3. D (See Table 1.) Figure 2 shows that fusion protein 219127 bound with high affinity to ferritin at pH 4.5.

[0105] TIFF2025501751000002.tif60170

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

[0107] Affinity chromatography with ferritin. For affinity chromatography, Praesto 85-219127 was packed into a superformance column housing (Gotec, 5-50) and equilibrated with 1xPBS pH 7.3. The resin was loaded with 30 mL of a solution containing ferritin (e.g. at least 500 μg human ferritin, Merck, Cat. No.: 341482) with a residence time of 6 min. A wash step with 100 mM sodium acetate pH 4.5 was also included to confirm that ferritin still binds the ligand at pH 4.5. Elution was performed with a gradient from pH 4.5 to pH 2.0 over 15 column volumes (CV) using 100 mM citric acid. The pH of the buffer fractions containing the target was determined.

[0108] Fusion protein 219127 (SEQ ID NO:6, which contains SEQ ID NO:2) showed a single peak homogenous elution profile (see FIG. 3A).

[0109] The maximum peak of the pH gradient profile was at pH 3.1. The eluted ferritin showed high purity by SDS-PAGE (Figure 3B). Highly enriched ferritin was observed compared to the initial load. The identity of ferritin in the eluted fractions was confirmed by a specific ferritin ELISA (IBL international; Catalog No: 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 1xPBS pH 7.3. Praesto85_219214, Praesto85_219126 or Praesto85_219127 purified human ferritin (Merck, Cat. No.: 341482, 1.5mg / ml, 400μl, PBS pH 7.3) were mixed with 20μl of resin for 1h at room temperature. The matrix was washed twice with 300μl 1xPBS pH 7.3 and bound proteins were eluted with 3x100μl 100mM citric acid pH 2.0. The amount of eluted ferritin was calculated by Ferritin ELISA (IBL international, Cat. No. 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, respectively.

[0112] array SEQ ID NO: 1: Ferritin binding protein AKFDXXQXYADXXILXLXNLTEEQRNAFRQSLSDDPSVSWXVLXEARXLNEXQAPK X may be any naturally occurring 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. SEQ ID NO: 2: Ferritin binding protein (218990) AKFDQWQSYADHQILKLPNLTEEQRNAFRQSLSDDPSVSWEVLWEARELNESQAPK SEQ ID NO: 3: Ferritin binding protein (218989) AKFDAYQSYADKEILQLPNLTEEQRNAFRQSLSDDPSVSWEVLQEARVLNESQAPK SEQ ID NO: 4: Ferritin binding protein (218894) AKFDQFQLYADKEILELFNLTEEQRNAFRQSLSDDPSVSWYVLWEARMLNEAQAPK SEQ ID NO:5: Fusion protein containing SEQ ID NO:2 (219126) AKFDQWQSYADHQILKLPNLTEEQRNAFRQSLSDDPSVSWEVLWEARELNESQAPKIAAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKIAAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKC SEQ ID NO:6: Fusion protein containing SEQ ID NO:2 (dimer) (219127) AKFDQWQSYADHQILKLPNLTEEQRNAFRQSLSDDPSVSWEVLWEARELNESQAPKIAAKFDQWQSYADHQILKLPNLTEEQRNAFRQSLSDDPSVSWEVLWEARELNESQAPKI AAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKIAAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKC SEQ ID NO: 7: Fusion protein containing SEQ ID NO: 3 (219124) AKFDAYQSYADKEILQLPNLTEEQRNAFRQSLSDDPSVSWEVLQEARVLNESQAPKIAAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKIAAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKC SEQ ID NO:8: Fusion protein containing SEQ ID NO:3 (dimer) (219125) AKFDAYQSYADKEILQLPNLTEEQRNAFRQSLSDDPSVSWEVLQEARVLNESQAPKIAAKFDAYQSYADKEILQLPNLTEEQRNAFRQSLSDDPSVSWEVLQEARVLNESQAPKI AAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKIAAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKC SEQ ID NO: 9: Fusion protein containing SEQ ID NO: 4 (219122) AKFDQFQLYADKEILELFNLTEEQRNAFRQSLSDDPSVSWYVLWEARMLNEAQAPKIAAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKIAAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKC SEQ ID NO:10: Fusion protein containing SEQ ID NO:4 (dimer) (219123) AKFDQFQLYADKEILELFNLTEEQRNAFRQSLSDDPSVSWYVLWEARMLNEAQAPKIAAKFDQFQLYADKEILELFNLTEEQRNAFRQSLSDDPSVSWYVLWEARMLNEAQAPKI AAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKIAAQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKC SEQ ID NO: 11: Non-ferritin binding protein AQHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPK SEQ ID NO: 12: Ferritin MTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLEFPSPISPSPSCWHHYTTNRPQPQHHLLRPRRRKRPHSIPTPILIFRSP

Claims

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

2. The binding protein of claim 1 , wherein 2, 3, 4, 5, or 6 binding proteins are linked to each other.

3. The binding protein according to 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 according to claim 1 and at least one non-ferritin binding protein.

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

6. A ferritin-binding protein according to any one of claims 1 to 3 or a fusion protein according to claims 4 to 5 for use in technical applications such as affinity chromatography.

7. An affinity separation matrix comprising the ferritin-binding protein according to any one of claims 1 to 3 or the fusion protein according to claims 4 to 5.

8. Use of the ferritin-binding protein according to any one of claims 1 to 3 or the fusion protein according to claims 4 to 5 or the affinity separation matrix according to claim 7 for affinity chromatography.

9. 1. A method for purifying ferritin, the method comprising: (i) providing a liquid containing ferritin; (ii) providing an affinity separation matrix comprising at least one ferritin-binding protein according to any one of claims 1 to 3 or a fusion protein according to claims 4 to 5 bound to said affinity separation matrix, (iii) contacting said affinity separation matrix with said liquid under conditions allowing binding of at least one ferritin-binding protein according to any one of claims 1 to 3 or a fusion protein according to claims 4 to 5; and (iv) eluting the ferritin from the affinity purification matrix.

10. Use of a ferritin-binding protein according to claims 1 to 3 or a fusion protein according to claims 4 to 5 or an affinity matrix according to claim 7 in a method for analyzing the presence or absence of ferritin.

11. 1. A method for analyzing the presence or absence of ferritin in a liquid sample, said method comprising the steps of: (i) providing a liquid containing ferritin; (ii) Providing a ferritin-binding protein according to claims 1 to 3 or a fusion protein according to claims 4 to 5, (iii) contacting the liquid of (i) with a ferritin-binding protein according to claims 1-3 or a fusion protein according to claims 4-5 under conditions allowing binding of said ferritin-binding protein; (iv) isolating the ferritin and binding protein complex; and (v) determining the amount of said ferritin binding protein in the liquid of (i); method.

12. A nucleic acid molecule encoding the ferritin-binding protein according to claims 1 to 3 or the fusion protein according to claims 4 to 5.

13. A vector comprising the nuclear molecule of claim 12.

14. A host cell or a non-human host comprising a ferritin-binding protein according to claims 1 to 3 or a fusion protein according to claims 4 to 5, a nucleic acid according to claim 12, and / or a vector according to claim 13.

15. A method for producing a ferritin-binding protein according to claims 1 to 3 or a fusion protein according to claims 4 to 5, comprising culturing a host cell according to claim 14 under suitable conditions to obtain a ferritin-binding protein or a fusion protein comprising said ferritin-binding protein, and optionally isolating said ferritin-binding protein or a fusion protein comprising said ferritin-binding protein.

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