Expression of truncated glial fibrillary acidic proteins

Abstract

The present invention relates to nucleic acids coding for a protein said protein comprising a truncated glial fibrillary acidic protein (GFAP), proteins comprising a truncated GFAP, expression constructs comprising a nucleic acid coding for a protein which comprises a truncated GFAP, vectors comprising the nucleic acids or expression constructs disclosed herein and eukaryotic cells comprising a nucleic acid, proteins, expression construct or vector disclosed herein. The present invention furthermore relates to compositions comprising the herein disclosed proteins and kits comprising the herein disclosed proteins and / or compositions. Furthermore, the present invention relates to the use of the herein disclosed proteins, compositions and / or kits for calibrating a measurement system for the in vitro quantification of GFAP or a variant thereof in a sample.

Description

[0001] EXPRESSION OF TRUNCATED GLIAL FIBRILLARY ACIDIC PROTEINS

[0002] Technical Field

[0003] The present invention relates to nucleic acids coding for a protein said protein comprising a truncated glial fibrillary acidic protein (GFAP), proteins comprising a truncated GFAP, expression constructs comprising a nucleic acid coding for a protein which comprises a truncated GFAP, vectors comprising the nucleic acids or expression constructs disclosed herein and eukaryotic cells comprising a nucleic acid, proteins, expression construct or vector disclosed herein. The present invention furthermore relates to methods for the production of the herein disclosed proteins. The present invention also relates to compositions comprising the herein disclosed proteins and kits comprising the herein disclosed proteins and / or compositions. Furthermore, the present invention relates to the use of the herein disclosed proteins, compositions and / or kits for calibrating a measurement system for the in vitro quantification of GFAP or a variant thereof in a sample.

[0004] Background

[0005] Glial fibrillary acidic protein (GFAP) is a type III intermediate filament. It is expressed by various cells in the central nervous system and is the predominantly expressed intermediate filament in mature astrocytes.

[0006] GFAP is used as a blood-derived biomarker in diagnosing and assessing traumatic brain or spinal cord injury as well as other diseases affecting the central nervous system, such as inflammatory diseases like multiple sclerosis or cancer like glioma. For example, the FDA approved a blood test for clinical use for the evaluation of mild traumatic brain injury in 2018.

[0007] For quantifying a protein with different assays, for example immunoassay, immuno sandwich assay or ELISA, a calibration of the assay is vital. For this, so called calibrators are used. Calibrators are protein standards employed at known concentrations to interpolate the concentration of a target protein in a sample. There are certain requirements for the use of a calibrator. One such requirement is that they have to be stable so that they can be stored for a longer time, e.g. as a lyophilisate. An unstable protein cannot be used as a calibrator, because the detectable amount of the protein will change significantly already after a brief storage and thus defeating the purpose of a calibrator with a known concentration.

[0008] Until now the only reliably available source of a GFAP calibrator is human brain tissue. This poses several issues: the supply depends on the availability of sufficient human brain tissue that also needs to be disease free. Furthermore, GFAP is purified from human brain tissue using urea, which is used to denature GFAP in order to keep it soluble. However, urea might interfere with subsequent uses in assays.

[0009] There is therefore a particular interest in obtaining a recombinantly expressed GFAP with the advantage that it will be safer and easier to obtain, and a more stable GFAP for use as a calibrator.

[0010] So far only prokaryotic expression systems have been tried to express GFAP. CN113005134A discloses the expression of a codon optimized GFAP for expression in A. coli. Full-length GFAP expressed in A. coli does not properly fold and inclusion bodies comprising GFAP are formed during expression. In order to purify GFAP from inclusion bodies further steps are necessary, which include denaturing the inclusion bodies and the proteins therein, and the refolding of the protein, which bears the risk that it is not properly refolded. Furthermore, the yield is much lower and various aggressive chemicals have to be employed. CN113005134A further discloses fusing a prokaryotic secretion signal to GFAP. However, purification is not performed from the cell culture medium, but by lysis of the cells, which means that the secretion was not successful and that the protein was not solubly expressed.

[0011] Quinlan et al. (Quinlan et al.. Expression in Escherichia coli of fragments of glial fibrillary acidic protein: characterization, assembly properties and paracrystal formation, Journal of Cell Science, 93, 71-83, 1989) disclose the expression of various fragments of mouse GFAP in A. coli. However, the fragments are again only present in inclusion bodies and have to be laboriously purified and refolded. Chen and Liem (Chen and Liem, The endless story of the glial fibrillary acidic protein, Journal of Cell Science, 107, 2299-2311, 1994) disclose the expression of GFAP deletion mutants with partial deletion mutants at the N- or C-terminus in human carcinoma cells to study the role of different parts of GFAP in intermediate fibre assembly. They found that only a part of the C-terminal tail domain of GFAP could be deleted without disturbing self-assembly, while the intact head domain at the N-terminus is required for self-assembly. They did not purify or analyse the stability of these mutants with respect to storage, in particular when lyophilised.

[0012] Therefore, there remains a need to produce a GFAP with an increased stability, in particular during lyophilisation for long-term storage. It would furthermore be beneficial to decrease costs and / or to ensure product usability over a long period of time.

[0013] Summary of the invention

[0014] In a first aspect, the present invention relates to a nucleic acid coding for a protein said protein comprising a truncated glial fibrillary acidic protein (GFAP), wherein the truncated GFAP comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 85 % sequence identity to SEQ ID NO: 2, wherein the protein does

[0015] (i) neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 10 consecutive amino acids, and / or

[0016] (ii) neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 10 consecutive amino acids.

[0017] The amino acid sequences of SEQ ID NO: 5 and 6 correspond to the amino acids 1 to 68 of full length GFAP of SEQ ID NO: 1 and the amino acids corresponding to amino acids 378 to 432 of full length GFAP of SEQ ID NO: 1, respectively.

[0018] The inventors have surprisingly found that specific deletions at the N- and / or C- terminus of GFAP lead to an increased stability of the truncated GFAP compared to the full-length protein, in particular when lyophilised. The increased stability is evidenced by the higher level of reconstitution of protein after lyophilisation as shown in the examples. The nucleic acids disclosed herein are particularly useful for expressing a protein comprising a truncated GFAP.

[0019] In a second aspect, the present invention relates to a protein comprising a truncated GFAP, wherein the truncated GFAP comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 85 % sequence identity to SEQ ID NO: 2, wherein the protein does

[0020] (i) neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 10 consecutive amino acids, and / or

[0021] (ii) neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 10 consecutive amino acids, .

[0022] In a fourth aspect, the present invention relates to an expression construct comprising a nucleic acid of the first.

[0023] In a fifth aspect, the present invention relates to a vector comprising an expression construct of the fourth aspect.

[0024] In a sixth aspect, the present invention relates to a eukaryotic cell comprising a nucleic acid of the first or third aspect, a protein of the second aspect, an expression construct of the fourth aspect or a vector of the fifth aspect.

[0025] In a seventh aspect, the present invention relates to a method for the production of a protein comprising a truncated GFAP comprising:

[0026] (a) providing a eukaryotic cell of the sixth aspect,

[0027] (b) culturing the eukaryotic cell in a cell culture medium under conditions that allow the cell to express the protein comprising a truncated GFAP encoded by a nucleic acid of the first or third aspect and thereby producing the protein comprising a truncated GFAP, and

[0028] (c) obtaining the protein comprising a truncated GFAP produced in step (b).

[0029] In an eighth aspect, the present invention relates to a protein comprising a truncated GFAP obtainable, in particular obtained, by a method of the seventh aspect.

[0030] In a ninth aspect, the present invention relates to a composition comprising a protein of the second or eighth aspect. In a tenth aspect, the present invention relates to a kit comprising a protein of the second or eighth aspect or at least one composition of the ninth aspect.

[0031] In an eleventh aspect, the present invention relates to a use of a protein of the second or eighth aspect, a composition of the ninth aspect or a kit of the tenth aspect for calibrating a measurement system for the in vitro quantification of GFAP or a variant thereof in a sample.

[0032] In a twelfth aspect, the present invention relates to a method for calibrating a measurement system for the in vitro quantification of GFAP or a variant thereof in a sample comprising a step of measuring at least once with the measurement system at least one composition according to the ninth aspect having a known concentration of the protein comprising a truncated GFAP.

[0033] List of figures

[0034] FIG. 1 A & B show SDS-PAGE analysis of Albumin(ALB)-GFAP-His8 (A) and ALB-GFAP_69-377-His8 (B) under reducing conditions:

[0035] (A) Line M : Marker SeeBlue™ Plus2 Pre-stained (Invitrogen), Line 1 : ALB-GFAP- Hiss

[0036] (B) Line M : Marker SeeBlue™ Plus2 Pre-stained (Invitrogen), Line 1 : high- molecular- weight impurities separated in the final purification step, Line 2: ALB- GFAP_69-377-His8, Line 3: low-molecular-weight impurities separated in final purification step.

[0037] FIG. 2 shows a SEC-MALS chromatogram (left axis: UV signal at 280 nm) received from the miniDAWN® TREOS® II. Optilab® T.rEX Detectors showed a molecular weight (right axis) of full length ALB-GFAP-Hiss.

[0038] FIG. 3 shows a SEC-MALS chromatogram (left axis: UV signal at 280 nm) received from the miniDAWN® TREOS® II. Optilab® T.rEX Detectors showed a molecular weight (right axis) of full length ALB-GFAP_67-377-His8.

[0039] FIG. 4 A & B show thermal unfolding traces recorded with Prometheus Panta System collected for ALB-GFAP-His8(A) and ALB-GFAP_69-377-His8(B). Detailed Description of the invention

[0040] Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular embodiments and examples described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0041] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedence.

[0042] In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0043] The following definitions and embodiments apply to the present disclosure in its entirety, especially to all aspects and embodiments of the invention.

[0044] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents, unless the content clearly dictates otherwise. The word "comprise", and variations such as "comprises" and "comprising", will be understood to imply 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. In some embodiments, the word “comprising” may include “consisting”.

[0045] The use of the alternative (e.g. “or”) should be understood to mean either one, both or any combination thereof of the alternatives.

[0046] The term “and / or” should be understood to mean either one, or both of the alternatives.

[0047] Percentages, concentrations, amounts and other numerical data may be expressed or presented herein in a “range” format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of "4 % to 20 %" should be interpreted to include not only the explicitly recited values of 4 % to 20 %, but to also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 4, 5, 6, 7, 8, 9, 10, . . . 18, 19, 20 % and sub-ranges such as from 4-10 %, 5-15 %, 10-20 %, etc. This same principle applies to ranges reciting minimal or maximal values. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.

[0048] As used herein and unless stated otherwise, it is to be understood that the term “about” is used synonymously with the term “approximately”. Illustratively and unless stated otherwise, the use of the term “about” when used in conjunction with a stated numerical value or range denotes somewhat more or somewhat less than the stated value or range, to within a range of ±15% of that stated, ±10% of that stated, ±5% of that stated, or conveniently ± 2% of that stated. Such values are thus encompassed by the scope of the claims reciting the terms “about” or “approximately”. As used in the present disclosure, "% w / v" refers to weight by volume percent, which is a unit of concentration measuring the amount of solute in grams (g) expressed as a percent of the total volume of solution in milliliters (ml).

[0049] As used in the present disclosure, "% v / v" refers to volume by volume percent, which is a unit of concentration measuring the amount of a specific solute in milliliters (ml) expressed as a percent of the total volume of solution in milliliters (ml).

[0050] The term "lyophilising" or "lyophilisation" refers to the freeze-drying of a substance by freezing it and then reducing the surrounding pressure (e.g. below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or less) to allow the frozen medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms "lyophilising" and "freeze-drying" are used herein interchangeably.

[0051] The term "reconstitute" relates to adding a solvent such as water to a dried product, such as a lyophilisate, to return it to a liquid state such as its original liquid state.

[0052] The term "recombinant" in the context of the present disclosure means "made through genetic engineering". In some embodiments, a "recombinant protein" in the context of the present disclosure is not occurring naturally.

[0053] The term “truncated” as used herein refers to a non-naturally occurring protein that differs from the naturally occurring protein, from which it is derived, by the deletion of at least one amino acid at one or both ends of the N- or C-terminus. A deletion within the sequence of a protein may not be considered as a truncation of a protein. Such a truncation can be created recombinantly or by using a peptidase. For example, the deletion of amino acids 1 to 20 at the N-terminus of a 100 amino acids long protein leads to a truncated protein lacking amino acids 1 to 20.

[0054] As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably herein and refer to temperatures from at least about 15 °C, e.g. from about 15 °C to about 35 °C, from about 15 °C to about 30 °C, from about 15 °C to about 25 °C or from about 17 °C to about 22 °C. Such temperatures will include 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 21 °C and 22 °C. The term "cryoprotectant" relates to a substance that is added to a formulation in order to protect the active ingredients during freezing.

[0055] The term "lyoprotectant" relates to a substance that is added to a formulation in order to protect the active ingredients during the freeze-drying.

[0056] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring. The term "found in nature" means "present in nature" and includes known objects as well as objects that have not yet been discovered and / or isolated from nature, but that may be discovered and / or isolated in the future from a natural source. The term “native” refers to a naturally occurring object.

[0057] According to the present invention, the term "peptide" refers to molecules which comprise about two or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150, consecutive amino acids linked to one another via peptide bonds. The term "polypeptide" refers to large peptides, in particular peptides having at least about 151 amino acids. "Peptides" and "polypeptides" are both protein molecules. Thus, the terms "peptide", "protein" and "polypeptide" are used herein usually as synonyms.

[0058] According to the present invention, a protein that “lacks the amino acids corresponding to amino acids 1 to 68 of SEQ ID NO: 1” refers to a protein that neither comprises amino acids 1 to 68 of SEQ ID NO: 1 nor parts thereof. Parts of amino acids 1 to 68 of SEQ ID NO: 1 may typically be at least 10, at least 9, at least 8, at least 7, at least 6, at least 5, at least 4 or at least 3 consecutive amino acids of amino acids 1 to 68 of SEQ ID NO: 1. Parts with a smaller minimal length are preferred. According to the present invention, a protein that “lacks the amino acids corresponding to amino acids 378 to 432 of SEQ ID NO: 1” refers to a protein that neither comprises amino acids 378 to 432 of SEQ ID NO: 1 nor parts thereof. Parts of amino acids 378 to 432 of SEQ ID NO: 1 may typically be at least 10, at least 9, at least 8, at least 7, at least 6, at least 5, at least 4 or at least 3 consecutive amino acids of amino acids 378 to 432 of SEQ ID NO: 1. Parts with a smaller minimal length are preferred. For example, the sequence S A A R R S Y V S S G E M, corresponding to the amino acids at position 8 to 20 of SEQ ID NO: 1, is a part of the amino acid sequence of SEQ ID NO: 1 that may be lacking from a protein according to the present invention.

[0059] According to the present invention, a protein that “does neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 10 consecutive amino acids” or a protein that “does neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 10 consecutive amino acids” refers to a protein in which, when a sequence alignment is performed, the alignment lacks all of the amino acids corresponding to the amino acids of the sequence in the referenced SEQ ID in their respective consecutive sequence and in case of a fragment it means that, when a sequence alignment to the protein is performed, the alignment lacks the amino acids of the fragment in their respective consecutive sequence. A fragment may typically be at least 10, at least 9, at least 8, at least 7, at least 6, at least 5, at least 4 or at least 3 consecutive amino acids of the referenced sequence. In the present invention, fragments with a smaller minimal length may be preferred. For example, the sequence S A A R R S Y V S S G E M, corresponding to the amino acids at position 8 to 20 of SEQ ID NO: 5, is a fragment that is of more than 10 consecutive amino acids that lacks from a protein according to the present invention that does neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 10 consecutive amino acids.

[0060] For the purposes of the present invention, "variants" of an amino acid sequence (peptide or polypeptide) may comprise amino acid insertion variants, amino acid addition variants, amino acid deletion variants and / or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, in particular those which are naturally occurring. The term "variant" includes, in particular, fragments of an amino acid sequence. Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid addition variants comprise amino- and / or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein. Amino acid deletion variants that comprise the deletion at the N-terminal and / or C-terminal end of the protein are also called N-terminal and / or C- terminal truncation variants. Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous peptides or polypeptides and / or to replacing amino acids with other ones having similar properties. In some embodiments, amino acid changes in peptide and polypeptide variants are conservative amino acid changes, i.e. substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In some embodiments, conservative amino acid substitutions include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0061] "Sequence similarity" indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences. The alignment for determining sequence similarity, such as sequence identity can be done with art known tools, such as using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS ::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0062] The terms "% identical" and "% identity" or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or "window of comparison", in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of algorithms, e.g. the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, the local homology algorithm by Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). Percent identity of two sequences may be determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website. The algorithm parameters used for BLASTN algorithm on the NCBI website may include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match / Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. The algorithm parameters used for BLASTP algorithm on the NCBI website may include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment.

[0063] Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g. the number of positions in the reference sequence) and multiplying this result by 100.

[0064] Throughout this disclosure, a higher sequence identity with reference to a specific sequence, such as a sequence of a specific SEQ ID NO, is preferred.

[0065] Homologous amino acid sequences exhibit according to the disclosure at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and, e.g. at least 95%, at least 98 or at least 99% identity of the amino acid residues.

[0066] The amino acid sequence variants described herein may readily be prepared by the skilled person, for example, by recombinant DNA manipulation. The manipulation of DNA sequences for preparing peptides or polypeptides having substitutions, additions, insertions or deletions, is described in detail in Molecular Cloning: A Laboratory Manual, 4th Edition, M.R. Green and J. Sambrook et al. (1989), eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012, for example. Furthermore, the peptides, polypeptides and amino acid variants described herein may be readily prepared with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis and similar methods. A fragment or variant of an amino acid sequence (peptide or polypeptide) may be a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e. it is functionally equivalent. With respect to sequences of binding agents such as antibodies, one particular function is one or more binding activities displayed by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant", as used herein, in particular refers to a variant molecule or sequence that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that is still capable of fulfilling one or more of the functions of the parent molecule or sequence, e.g. binding to a target molecule. The modifications in the amino acid sequence of the parent molecule or sequence may not significantly affect or alter the characteristics of the molecule or sequence. The function of the functional fragment or functional variant may be reduced but still significantly present, e.g. function of the functional fragment or functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence. However, function of the functional fragment or functional variant may alternatively be enhanced compared to the parent molecule or sequence.

[0067] According to various embodiments of the present disclosure, a nucleic acid encoding a peptide or polypeptide is taken up by or introduced, i.e. transfected or transduced, into a cell which cell may be present in vitro or in a subject, resulting in expression of said peptide or polypeptide. The cell may, e.g. express the encoded peptide or polypeptide intracellularly (e.g. in the cytoplasm and / or in the nucleus), may secrete the encoded peptide or polypeptide, and / or may express it on the surface. According to the present disclosure, terms such as "nucleic acid expressing" and "nucleic acid encoding" or similar terms are used interchangeably herein and with respect to a particular peptide or polypeptide mean that the nucleic acid, if present in the appropriate environment, e.g. within a cell, can be expressed to produce said peptide or polypeptide.

[0068] The term "expression" as used herein includes the transcription and / or translation of a particular nucleotide sequence.

[0069] In the context of the present disclosure, the term "transcription" relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA (especially mRNA). Subsequently, the RNA may be translated into peptide or polypeptide.

[0070] With respect to RNA, the term "expression" or "translation" relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or polypeptide.

[0071] The term "polydispersity index" (£>M) refers to a measure of the heterogeneity of sizes of molecules or particles in a mixture. A collection of objects is called uniform if the objects have the same size, shape, or mass. A sample of objects that have an inconsistent size, shape and mass distribution is called non-uniform. The polydispersity index can be calculated using the equation DM = Mw / Mn, where Afwis the weight-average molecular weight and Mnis the number-average molecular weight.

[0072] The "polydispersity index" may be calculated by a method known to the skilled person, in particular it may be calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the "average diameter". Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of nanoparticles.

[0073] The "hydrodynamic radius" (which is sometimes called "Stokes radius" or "Stokes- Einstein radius") of a particle is the radius of a hypothetical hard sphere that diffuses at the same rate as said particle (plural hydrodynamic radii). The hydrodynamic radius is related to the mobility of the particle, taking into account not only size but also solvent effects. For example, a smaller charged particle with stronger hydration may have a greater hydrodynamic radius than a larger charged particle with weaker hydration. This is because the smaller particle drags a greater number of water molecules with it as it moves through the solution. Since the actual dimensions of the particle in a solvent are not directly measurable, the hydrodynamic radius may be defined by the Stokes-Einstein equation: wherein fe is the Boltzmann constant; T is the temperature; / is the viscosity of the solvent; and D is the diffusion coefficient. The diffusion coefficient can be determined experimentally, e.g. by using dynamic light scattering (DLS). Thus, one procedure to determine the hydrodynamic radius of a particle or a population of particles (such as the hydrodynamic radius of particles contained in a sample or control composition as disclosed herein or the hydrodynamic radius of a particle peak obtained from subjecting such a sample or control composition to field-flow fractionation) is to measure the DLS signal of said particle or population of particles (such as DLS signal of particles contained in a sample or control composition as disclosed herein or the DLS signal of a particle peak obtained from subjecting such a sample or control composition to field-flow fractionation). The expression "light scattering" as used herein refers to the physical process where light is forced to deviate from a straight trajectory by one or more paths due to localized non- uniformities in the medium through which the light passes.

[0074] The term "UV" means ultraviolet and designates a band of the electromagnetic spectrum with a wavelength from 10 nm to 400 nm, i.e. shorter than that of visible light but longer than X- rays.

[0075] The expression "multi-angle light scattering" or "MALS" as used herein relates to a technique for measuring the light scattered by a sample into a plurality of angles. "Multi-angle" means in this respect that scattered light can be detected at different discrete angles as measured, for example, by a single detector moved over a range including the specific angles selected or an array of detectors fixed at specific angular locations. In certain embodiments, the light source used in MALS is a laser source (MALLS: multi-angle laser light scattering). Based on the MALS signal of a composition comprising particles and by using an appropriate formalism (e.g. Zimm plot, Berry plot, or Debye plot), it is possible to determine the radius of gyration (Rg) and, thus, the size of said particles (see, e.g., Buchholz et al. (Electrophoresis 22 (2001), 4118-4128); B.H. Zimm (J. Chem. Phys. 13 (1945), 141; P. Debye (J. Appl. Phys. 15 (1944): 338; and W. Burchard (Anal. Chem. 75 (2003), 4279-4291).

[0076] The expression "dynamic light scattering" or "DLS" as used herein refers to a technique to determine the size and size distribution profile of particles, in particular with respect to the hydrodynamic radius of the particles. A monochromatic light source, usually a laser, is shot through a polarizer and into a sample. The scattered light then goes through a second polarizer where it is detected and the resulting image is projected onto a screen. The particles in the solution are being hit with the light and diffract the light in all directions. The diffracted light from the particles can either interfere constructively (light regions) or destructively (dark regions). This process is repeated at short time intervals and the resulting set of speckle patterns are analyzed by an autocorrelator that compares the intensity of light at each spot over time. The expression "static light scattering" or "SLS" as used herein refers to a technique to determine the size and size distribution profile of particles, in particular with respect to the radius of gyration of the particles, and / or the molar mass of particles. A high-intensity monochromatic light, usually a laser, is launched in a solution containing the particles. One or many detectors are used to measure the scattering intensity at one or many angles. The angular dependence is needed to obtain accurate measurements of both molar mass and size for all macromolecules of radius. Hence simultaneous measurements at several angles relative to the direction of incident light, known as multi-angle light scattering (MALS) or multi-angle laser light scattering (MALLS), is generally regarded as the standard implementation of static light scattering.

[0077] The term "C-terminus" refers to the carboxyl-terminus of a peptide chain. It is also called “carboxy-terminus” or “COOH-terminus”. A peptide chain comprises amino acids connected to one another via peptide bonds involving the amine group at the Caof the amino acid and the carboxyl-group. This forms a chain of amino acids connected via the amine group of X+l amino acid and carboxyl-group of the X amino acid. As the amino acids at both ends are not further connected to another amino acid this leaves the amine group at one end and the carboxyl group at the other end free. The C-terminus is considered to be the end of the amino acid chain and the N-terminus the start of the chain. Amino acid sequences are therefore denoted starting from the amino acid with the free amine group at Cato the amino acid with the free carboxyl group at the Ca.

[0078] The term "N-terminus" refers to amine-terminus of a peptide chain. It is also called “amino-terminus”, “amine-terminus” or “NH2-terminus”. The N-terminus is considered to be the start of the amino acid chain.

[0079] The term "calibrator" refers to a protein used as a protein standard at known concentrations to interpolate the concentration of a target protein in a sample and / or to recalibrate a measurement system. After a standard curve has been established using a calibrator at different concentrations, a measurement system can be recalibrated by the use of one or two compositions comprising the calibrator at a known concentration. By measuring a composition with a known concentration the skilled person is able to correlate the output signal from a measurement system to a particular concentration and in case a master standard curve has previously been calculated for this measurement system, often a single point recalibration suffices to confidently quantify an analyte within a sample over the whole range the previous master standard curve has been established for.

[0080] The term "tag" refers to an amino acid sequence that is fused with a protein recombinantly. Tags can be used for various purposes such as purifying a protein or detected it in situ. Some tags can even serve two different purposes, such as maltose binding protein-tag, which can be used as a solubilisation enhancer and as an affinity tag for purification. Tags can be fused to a peptide or protein at the N-terminus or C- terminus or can be located within an amino acid sequence. A protein can have more than one tag, e.g. two affinity tags for two different purification steps or for different purposes such as detection and purification. The terms "tag", "protein tag" and "peptide tag" are used herein as synonyms. Examples of protein tags include His-tag, FLAG-tag, HA(hemagglutinin)-tag, Avi-tag, strep-tag, MBP(maltose binding protein)-tag, Fc-tag, GFP(green fluorescent protein)-tag or GST(glutatione S transferase)-tag.

[0081] An Fc-tag is a protein tag comprising at least a part of the crystallizable fragment (Fc) of an immunoglobulin G (IgG). It can be fused recombinantly to a peptide sequence and the fusion protein can subsequently be purified via Protein A or Protein G affinity chromatography. The affinity of Protein A and Protein G depend on the species and class of IgG from which the Fc-tag is derived.

[0082] The term "eukaryotic secretion tag" refers to a tag that can be fused recombinantly to a protein's amino acid sequence and that leads to the secretion of the protein from a eukaryotic cell. The eukaryotic secretion tag usually comprises a signal peptide derived from a eukaryotic preprotein, but can also comprise further parts of the preprotein, such as further amino acids not part of the signal peptide or further protein domains. In some embodiments, a eukaryotic secretion tag can also comprise of a complete amino acid sequence of a preprotein. "Eukaryotic secretory protein" refers to a protein that is secreted by a eukaryotic cell. A eukaryotic secretory protein is secreted by a eukaryotic cell. Secretion by a eukaryotic cell usually includes the cleavage of the signal peptide during translocation into the endoplasmatic reticulum. A eukaryotic secretory protein lacks therefore a signal peptide. Furthermore, eukaryotic secretory proteins can have further post-translational modifications, such as glycosylations and / or the cleavage of further parts of the protein. The term “eukaryotic secretory protein” refers in particular to the processed protein and not to the preprotein or preproprotein.

[0083] "Preprotein" refers to a protein precursor, in particular of a secretory protein, comprising a signal peptide, also called pre-sequence. Preproteins are secreted from a cell. In eukaryotic cells the signal peptide is usually cleaved off during translocation of the polypeptide chain of the preprotein into the endoplasmatic reticulum or after translocation. With the cleavage of the signal peptide the preprotein ceases to exist. The protein is then further processed in the ER and optionally in the golgi apparatus and are ultimately send to the cell membrane, where secretory proteins are then secreted.

[0084] The term “signal peptide” refers to an amino acid sequence of 14 to 35 amino acids that is generally located at the N-terminus of a eukaryotic preprotein that is secreted by a cell. The core of the signal peptide is commonly a hydrophobic stretch of amino acids that is usually 5 to 16 amino acids long. This hydrophobic stretch has the tendency to form an a-helix. Towards the C-terminal end of the signal peptide, after the hydrophobic stretch and before the amino acid sequence of the (pro)protein starts, is usually a protease cleavage site for the signal peptidase. The signal peptide usually serves as the recognition signal for the signal-recognition particle (SPR), which usually binds to the signal peptide as it emerges from the ribosome during translation, halts further translation and is subsequently bound by the SRP receptor at the endoplasmatic reticulum (ER). The nascent protein chain is than translocated into the ER during translation. Either during translocation or thereafter, the signal peptide is cleaved off the (pro)protein by the signal peptidase.

[0085] The term "fusion protein" refers to a polypeptide or protein comprising two or more subunits. Preferably, the fusion protein is a translational fusion between the two or more subunits. The translational fusion may be generated by genetically engineering the coding nucleotide sequence for one subunit in a reading frame with the coding nucleotide sequence of a further subunit. Subunits may be interspersed by a linker.

[0086] The term "expression construct" refers to a nucleic acid comprising a coding sequence and further nucleotide sequences operatively linked to the coding sequence that are needed for the expression of the coding sequence. While a promoter and a terminator are required for efficiently starting and terminating translation of the coding sequence into mRNA, other sequences, such as 5' or 3' UTRs or enhancers are optional, but can aid expression of the coding sequence.

[0087] The term "isolated" means a protein or polypeptide, nucleic acid, cell, or other specified material or component that has been separated from at least one other material or component, including but not limited to, other proteins, nucleic acids, cells, etc. An isolated protein or polypeptide, nucleic acid, cell or other specified material or component may typically be in a form that does not occur in nature. For example, an isolated nucleic acid may be a nucleic acid that has been isolated from a cell, in which it originally was present.

[0088] The amount, concentration or level of a protein in a composition, such as a solution, may be measured by different methods known to the skilled person. Such methods may include mass spectrometry, UV absorbance, Bradford assay, Lowry assay or an immuno assay, such as ELISA. The percentage of a specific reconstituted protein from a lyophilisate may be determined by measuring the amount or concentration of the protein in the solution obtained by reconstitution and comparing this amount or concentration with the amount or concentration of the protein in the solution used to obtain the lyophilisate. When using concentration, the solution used to obtain the lyophilisate and the solution obtained by reconstitution have to have the same volume or the concentration values have to be adjusted to the same volume. For example, a lyophilisate X was produced using 500 pL of a solution comprising 2 mg / mL of protein Y. The lyophilisate was reconstituted to 1 mL and the concentration of protein Y in the resolubilized solution was 0.5 mg / mL. The adjusted concentration in the resolubilized solution is 1 mg / mL and the percentage of reconstituted protein is 50 % = (1 mg / mL 2 mg / mL) x 100. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0089] In a first aspect, the present invention relates to a nucleic acid coding for a protein said protein comprising a truncated glial fibrillary acidic protein (GFAP), wherein the truncated GFAP comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 85 % sequence identity to SEQ ID NO: 2, wherein the protein does

[0090] (i) neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 10 consecutive amino acids, and / or

[0091] (ii) neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 10 consecutive amino acids.

[0092] The amino acid sequences of SEQ ID NO: 5 and 6 correspond to the amino acids 1 to 68 of full length GFAP of SEQ ID NO: 1 and the amino acids corresponding to amino acids 378 to 432 of full length GFAP of SEQ ID NO: 1, respectively. A nucleic acid according to the present invention may therefore also be a nucleic acid coding for a protein said protein comprising a truncated glial fibrillary acidic protein (GFAP), wherein the truncated GFAP comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 85 % sequence identity to SEQ ID NO: 2, wherein the protein lacks the amino acids corresponding to amino acids 1 to 68 of SEQ ID NO: 1 and / or the amino acids corresponding to amino acids 378 to 432 of SEQ ID NO: 1.

[0093] In some embodiments of the present invention, the nucleic acid is DNA or RNA, in particular mRNA.

[0094] In specific embodiments of the present invention, the nucleic acid is DNA.

[0095] In some embodiments of the present invention, the nucleic acid is an isolated nucleic acid.

[0096] In some embodiments of the present invention, the amino acid sequence of the truncated GFAP is derived from a human GFAP. In some embodiments of the present invention, the amino acid sequence of the GFAP is derived from a human GFAP identified by UniProt accession number P14136.

[0097] In some embodiments of the present invention, the protein does neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 5 consecutive amino acids, and / or neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 5 consecutive amino acids.

[0098] In some embodiments of the present invention, the protein does neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 3 consecutive amino acids, and / or neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 3 consecutive amino acids.

[0099] In some embodiments of the present invention, the protein does neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 10 consecutive amino acids, and neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 10 consecutive amino acids.

[0100] In some embodiments of the present invention, the protein does neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 5 consecutive amino acids, and neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 5 consecutive amino acids.

[0101] In some embodiments of the present invention, the protein does neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 3 consecutive amino acids, and neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 3 consecutive amino acids.

[0102] In some embodiments of the present invention, the protein does not comprise a fragment of at least 9, at least 8, at least 7, at least 6, at least 5, at least 4 or at least 3 consecutive amino acids of

[0103] (i) SEQ ID NO: 5 and / or

[0104] (ii) SEQ ID NO: 6.

[0105] In some embodiments of the present invention, the amino acid sequence of the truncated GFAP comprises or consists of SEQ ID NO: 3, SEQ ID NO: 4 or a variant thereof having at least 85 %, at least 90 % or at least 95 % sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4.

[0106] In some embodiments of the present invention, the amino acid sequence of the truncated GFAP comprises or consists of SEQ ID NO: 3 or SEQ ID NO: 4.

[0107] In some embodiments of the present invention, the amino acid sequence of the truncated GFAP consists of SEQ ID NO: 3, SEQ ID NO: 4 or a variant thereof having at least 85 %, at least 90 % or at least 95 % sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4.

[0108] In some embodiments of the present invention, the amino acid sequence of the truncated GFAP consists of SEQ ID NO: 3 or SEQ ID NO: 4.

[0109] In some embodiments of the present invention, the truncated GFAP comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 90 % or at least 95 % sequence identity to SEQ ID NO: 2.

[0110] In some embodiments of the present invention, the truncated GFAP comprises the amino acid sequence of SEQ ID NO: 2.

[0111] In some embodiments of the present invention, the amino acid sequence of the truncated GFAP consists of SEQ ID NO: 2 or a variant thereof having at least 85 %, at least 90 % or at least 95 % sequence identity to SEQ ID NO: 2.

[0112] In some embodiments of the present invention, the amino acid sequence of the truncated GFAP consists of SEQ ID NO: 2.

[0113] In some embodiments of the present invention, the first aspect of the present invention relates to a nucleic acid coding for a protein said protein comprising a truncated glial fibrillary acidic protein (GFAP), wherein the truncated GFAP comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 85 %, at least 90 % or at least 95 % sequence identity to SEQ ID NO: 2, wherein the protein does

[0114] (i) neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 10 consecutive amino acids, and / or (ii) neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 10 consecutive amino acids.

[0115] In some embodiments of the present invention, the first aspect of the present invention relates to a nucleic acid coding for a protein said protein comprising a truncated glial fibrillary acidic protein (GFAP), wherein the truncated GFAP comprises the amino acid sequence of SEQ ID NO: 2, wherein the protein does

[0116] (i) neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 10 consecutive amino acids, and / or

[0117] (ii) neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 10 consecutive amino acids.

[0118] In some embodiments of the present invention, the first aspect of the present invention relates to a nucleic acid coding for a protein said protein comprising a truncated glial fibrillary acidic protein (GFAP), wherein the truncated GFAP comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 85 %, at least 90 % or at least 95 % sequence identity to SEQ ID NO: 2, wherein the protein does

[0119] (i) neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 10 consecutive amino acids, and

[0120] (ii) neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 10 consecutive amino acids.

[0121] In some embodiments of the present invention, the first aspect of the present invention relates to a nucleic acid coding for a protein said protein comprising a truncated glial fibrillary acidic protein (GFAP), wherein the truncated GFAP comprises the amino acid sequence of SEQ ID NO: 2, wherein the protein does

[0122] (i) neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 10 consecutive amino acids, and

[0123] (ii) neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 10 consecutive amino acids. In some embodiments of the present invention, the first aspect of the present invention relates to a nucleic acid coding for a protein said protein comprising a truncated glial fibrillary acidic protein (GFAP), wherein the truncated GFAP consists of the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 85 %, at least 90 % or at least 95 % sequence identity to SEQ ID NO: 2, wherein the protein does

[0124] (i) neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 10 consecutive amino acids, and / or

[0125] (ii) neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 10 consecutive amino acids.

[0126] In some embodiments of the present invention, the first aspect of the present invention relates to a nucleic acid coding for a protein said protein comprising a truncated glial fibrillary acidic protein (GFAP), wherein the truncated GFAP consists of the amino acid sequence of SEQ ID NO: 2, wherein the protein does

[0127] (i) neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 10 consecutive amino acids, and / or

[0128] (ii) neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 10 consecutive amino acids.

[0129] In some embodiments of the present invention, the first aspect of the present invention relates to a nucleic acid coding for a protein said protein comprising a truncated glial fibrillary acidic protein (GFAP), wherein the truncated GFAP consists of the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 85 %, at least 90 % or at least 95 % sequence identity to SEQ ID NO: 2, wherein the protein does

[0130] (i) neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 10 consecutive amino acids, and

[0131] (ii) neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 10 consecutive amino acids.

[0132] In some embodiments of the present invention, the first aspect of the present invention relates to a nucleic acid coding for a protein said protein comprising a truncated glial fibrillary acidic protein (GFAP), wherein the truncated GFAP consists of the amino acid sequence of SEQ ID NO: 2, wherein the protein does

[0133] (i) neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 10 consecutive amino acids, and

[0134] (ii) neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 10 consecutive amino acids.

[0135] The protein of the present invention may be a fusion protein and may comprise, further to the truncated GFAP, a protein tag. This tag may help in purification and / or increase solubility and / or yield.

[0136] In some embodiments of the present invention, the protein further comprises at least one tag.

[0137] In some embodiments of the present invention, at least one tag is a fluorescent tag, an affinity tag, solubility enhancing tag and / or eukaryotic secretion tag.

[0138] In some embodiments of the present invention, at least one tag is present at the N- terminus or C-terminus of the protein.

[0139] In some embodiments of the present invention, a linker sequence is comprised between the at least one tag and the truncated GFAP. In some embodiments of the present invention, a linker sequence is comprised between any further tag and the truncated GFAP.

[0140] In some embodiments of the present invention, at least one linker sequence comprises a protease cleavage site.

[0141] A linker as used according to the present invention may contain a protease cleavage site, e.g. a furin cleavage site. The advantage is that any tag fused via a linker with a protease cleavage site can be removed, for example during or after purification, and may therefore not interfere with subsequent applications or storage of the protein.

[0142] In some embodiments of the present invention, the protease cleavage site is a TEV protease cleavage site, a furin cleavage site, IgA protease cleavage site, a SUMO protease cleavage site, a NEDD8 protease cleavage site, a ubiquitin protease cleavage site, a Thrombin cleavage site, a Factor Xa cleavage site or a HRV 3C protease cleavage site.

[0143] In some embodiments of the present invention, at least one linker sequence comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and amino acid sequences having at least 85 %, at least 90 % or at least 95 % sequence identity to any one of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. The linker sequence of SEQ ID NO: 11 comprises a furin cleavage site. In an embodiment, the amino acid sequences having at least 85 %, at least 90 % or at least 95 % sequence identity to SEQ ID NO: 11 comprise a functional furin cleavage site, i.e. a furin cleavage site that can be cleaved by furin.

[0144] In some embodiments of the present invention, at least one linker sequence comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: H and SEQ ID NO: 12.

[0145] In some embodiments of the present invention, at least one linker sequence consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and amino acid sequences having at least 85 %, at least 90 % or at least 95 % sequence identity to any one of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. The linker sequence of SEQ ID NO: 11 comprises a furin cleavage site. In an embodiment, the amino acid sequences having at least 85 %, at least 90 % or at least 95 % sequence identity to SEQ ID NO: 11 comprise a functional furin cleavage site, i.e. a furin cleavage site that can be cleaved by furin.

[0146] In some embodiments of the present invention, at least one linker sequence consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

[0147] In some embodiments of the present invention, the tag is a eukaryotic secretion tag.

[0148] The eukaryotic secretion tag may provide the advantage that the protein is secreted into the supernatant, i.e. the cell culture medium, and thus can be easier purified, because the cell already separates proteins that are not correctly folded and may aggregate. The eukaryotic secretion tag may provide more advantages than secretion to the cell culture medium. Depending in particular on the type of eukaryotic secretion tag, it may also increase solubility and / or can be used for further purification subsequent to secretion. It is preferred to fuse a eukaryotic secretion tag to the N-terminus of the protein, because typically the signal recognition particle, responsible for guiding the ribosome and nascent peptide chain to the ER, binds to the signal peptide at the N-terminus of a eukaryotic secretory protein. Depending on the signal peptide, the eukaryotic secretion tag may be comprised at the N-terminus of the protein.

[0149] In some embodiments of the present invention, the eukaryotic secretion tag comprises a signal peptide from a preprotein of a eukaryotic secretory protein.

[0150] In some embodiments of the present invention, the eukaryotic secretion tag consists of a signal peptide from a preprotein of a eukaryotic secretory protein.

[0151] In some embodiments of the present invention, the eukaryotic secretion tag is a preprotein of a eukaryotic secretory protein or part thereof, wherein the fragment comprises the signal peptide and at least one further amino acid.

[0152] In some embodiments of the present invention, the eukaryotic secretion tag comprises the insulin signal peptide, albumin domain 1 (ADI domain), albumin or serotransferrin. In some embodiments of the present invention, the eukaryotic secretion tag is selected from the group consisting of the insulin signal peptide, albumin domain 1 (ADI domain), albumin and serotransferrin.

[0153] In some embodiments of the present invention, the eukaryotic secretion tag is derived from a mammalian protein. In some embodiments of the present invention, the eukaryotic secretion tag is derived from a mouse, rat, goat, enopus laevis or a human protein. Typically, the eukaryotic secretion tag is derived from a human protein.

[0154] In some embodiments of the invention, the eukaryotic secretion tag comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and amino acid sequences having at least 85 %, at least 90 % or at least 95 % sequence identity to any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

[0155] In some embodiments of the present invention, the eukaryotic secretion tag comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

[0156] In some embodiments of the invention, the eukaryotic secretion tag consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and amino acid sequences having at least 85 %, at least 90 % or at least 95 % sequence identity to any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

[0157] In some embodiments of the present invention, the eukaryotic secretion tag consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

[0158] In some embodiments of the present invention, the protein comprises the eukaryotic secretion tag at its N-terminus.

[0159] The protein of the present invention may comprise, in addition to the eukaryotic secretion tag, an additional tag that may increase the solubility, e.g. MBP(maltose binding protein)-tag, and / or can be used for affinity chromatography as a further purification step, e.g. MBP- or His-tag.

[0160] In some embodiments of the present invention, the protein further comprises a eukaryotic secretion tag and a further tag.

[0161] In some embodiments of the present invention, at least one further tag is N-terminal or C-terminal of the GFAP.

[0162] In some embodiments of the present invention, the protein further comprises a eukaryotic secretion tag at its N-terminus and an affinity tag at its C-terminus.

[0163] In some embodiments of the present invention, the protein further comprises a eukaryotic secretion tag at its N-terminus and a His-tag at its C-terminus. In some embodiments of the present invention, a linker sequence is comprised between the eukaryotic secretion tag and the truncated GFAP and optionally between the further tag and the truncated GFAP.

[0164] In some embodiments of the present invention, a linker sequence is comprised between the eukaryotic secretion tag and the truncated GFAP and between the further tag and the truncated GFAP, wherein the further tag is an affinity-tag, typically a His-tag.

[0165] In some embodiments of the present invention, the protein comprises or consists of the amino acid sequence of SEQ ID NO: 18 or a variant thereof having at least 85 %, at least 90 % or at least 95 % sequence identity to SEQ ID NO: 18.

[0166] In some embodiments of the present invention, the protein comprises or consists of the amino acid sequence of SEQ ID NO: 18.

[0167] In some embodiments of the present invention, the protein consists of the amino acid sequence of SEQ ID NO: 18 or a variant thereof having at least 85 %, at least 90 % or at least 95 % sequence identity to SEQ ID NO: 18.

[0168] In some embodiments of the present invention, the protein consists of the amino acid sequence of SEQ ID NO: 18.

[0169] In a second aspect, the present invention relates to a protein comprising a truncated GFAP, wherein the truncated GFAP comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 85 % sequence identity to SEQ ID NO: 2, wherein the protein does

[0170] (i) neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 10 consecutive amino acids, and / or

[0171] (ii) neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 10 consecutive amino acids.

[0172] All embodiments mentioned for the first aspect of the invention apply for the second aspect of the invention and vice versa. In some embodiments of the present invention, the protein is a recombinant protein.

[0173] In some embodiments of the present invention, the protein is an isolated protein.

[0174] In some embodiments of the present invention, the protein further comprises at least a part of a eukaryotic secretory protein.

[0175] In some embodiments of the present invention, the protein comprises a part of a eukaryotic secretory protein.

[0176] In some embodiments of the present invention, the part of a eukaryotic secretory protein is a domain, i.e. a protein domain, of a eukaryotic secretory protein. In some embodiments of the present invention, the domain is a functional domain of a eukaryotic secretory protein that exerts the function the domain typically exerts in the context of the full length eukaryotic secretory protein.

[0177] In some embodiments of the present invention, the eukaryotic secretory protein is a full length eukaryotic secretory protein.

[0178] The protein of the second aspect and the part of the eukaryotic secretory protein therein typically lack any signal peptide of a eukaryotic preprotein, because the signal peptide is cleaved off during secretion. A specific example of a part of a eukaryotic secretory protein is the albumin domain 1.

[0179] In some embodiments of the present invention, the eukaryotic secretory protein is selected from the group consisting of the albumin domain 1 (ADI domain), albumin and serotransferrin.

[0180] In some embodiments of the present invention, the eukaryotic secretory protein or part thereof is derived from a mammalian protein. In some embodiments of the present invention, the eukaryotic secretory protein or part thereof is derived from a mouse, rat, goat, enopus laevis or human eukaryotic secretory protein. Typically, the amino acid sequence of the eukaryotic secretory protein or part thereof is derived from a human eukaryotic secretory protein.

[0181] In some embodiments of the present invention, the eukaryotic secretory protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and sequences having at least 85 %, at least 90 % or at least 95 % sequence identity to any one of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0182] In some embodiments of the present invention, the eukaryotic secretory protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

[0183] In some embodiments of the present invention, the eukaryotic secretory protein consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and sequences having at least 85 %, at least 90 % or at least 95 % sequence identity to any one of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0184] In some embodiments of the present invention, the eukaryotic secretory protein consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

[0185] In some embodiments of the present invention, the protein comprises the eukaryotic secretory protein or part thereof at its N-terminus.

[0186] In some embodiments of the present invention, the protein of the second aspect further comprises at least a part of a eukaryotic secretory protein and a tag.

[0187] In some embodiments of the present invention, the protein of the second aspect further comprises at least a part of a eukaryotic secretory protein at the N-terminus and an affinity tag at its C-terminus.

[0188] In some embodiments of the present invention, the protein of the second aspect further comprises at least a part of a eukaryotic secretory protein at the N-terminus and a His-tag at its C-terminus.

[0189] In some embodiments of the present invention, a linker sequence is comprised between the eukaryotic secretory protein or part thereof and the truncated GFAP, and / or at least one tag and the truncated GFAP. In some embodiments of the present invention, a linker sequence is comprised between the eukaryotic secretory protein or part thereof, and the truncated GFAP and between the tag and the truncated GFAP.

[0190] In some embodiments of the present invention, a linker sequence is comprised between the eukaryotic secretory protein and the truncated GFAP, and between the tag and the truncated GFAP, wherein the tag is an affinity-tag, typically a His-tag.

[0191] In some embodiments of the present invention, the protein comprising a truncated GFAP has a polydispersity index of at most 1.1 when in solution. In some embodiments of the present invention, the protein comprising a truncated GFAP has a polydispersity index of at most 1.05 when in solution. In some embodiments of the present invention, the protein comprising a truncated GFAP has a polydispersity index of at most 1.01 when in solution.

[0192] The polydispersity index may be measured by a method known to the skilled person. In particular, the polydispersity index may be measured using MALS (Multi angle static light scattering). In some embodiments of the present invention, the light scattering instrument may be coupled to a size exclusion chromatography.

[0193] In some embodiments of the present invention, the solution in which the polydispersity index of the protein is measured is an aqueous solution. Typically, a solution comprising a buffering agent, salt(s) and / or further ingredients, such as nonionic surfactant, chelating agent or sodium azide, is used.

[0194] A specific method for measuring the polydispersity index is disclosed in the appended Examples.

[0195] In some embodiments of the present invention, the protein comprises or consists of the amino acid sequence of SEQ ID NO: 17 or a variant thereof having at least 85 %, at least 90 % or at least 95 % sequence identity to SEQ ID NO: 17.

[0196] In some embodiments of the present invention, the protein comprises or consists of the amino acid sequence of SEQ ID NO: 17. In some embodiments of the present invention, the protein consists of the amino acid sequence of SEQ ID NO: 17 or a variant thereof having at least 85 %, at least 90 % or at least 95 % sequence identity to SEQ ID NO: 17.

[0197] In some embodiments of the present invention, the protein consists of the amino acid sequence of SEQ ID NO: 17.

[0198] In a third aspect, the present invention relates to a nucleic acid coding for a protein of the second aspect.

[0199] All embodiments mentioned for the first and / or second aspect of the invention apply for the third aspect of the invention mutatis mutandis.

[0200] In a fourth aspect, the present invention relates to an expression construct comprising a nucleic acid of the first or third aspect.

[0201] All embodiments mentioned for the first, second and / or third aspect of the invention apply for the fourth aspect of the invention mutatis mutandis.

[0202] In some embodiments of the present invention, the expression construct of the present invention further comprises a promoter, a terminator, a 5' UTR and / or a 3' UTR. The promoter may be a eukaryotic promoter. In some embodiments of the present invention, the expression construct may further comprise an enhancer that enhances expression. The promoter, terminator, enhancer, 5’ UTR and / or 3’ UTR may be operably linked to the nucleic acid of the present invention comprised in the expression construct.

[0203] In some embodiments of the present invention, the promoter is an inducible promoter. Examples of such an inducible promoter include a lac promoter or tet (tetracycline) promoter with the tetracycline responsive element.

[0204] In some embodiments of the present invention, the promoter is a constitutive promoter. An example of such a constitutive promoter is the cytomegalovirus (CMV) promoter.

[0205] In some embodiments of the present invention, the expression construct comprises the human cytomegalovirus immediate-early enhancer / promoter region. In some embodiments of the present invention, the expression construct comprises the bovine growth hormone polyadenylation signal.

[0206] In a fifth aspect, the present invention relates to vector comprising an expression construct of the fourth aspect.

[0207] All embodiments mentioned for the first, second, third and / or fourth aspect of the invention apply for the fifth aspect of the invention mutatis mutandis.

[0208] In some embodiments of the present invention, the vector of the present invention further comprises at least one selection marker and / or at least one origin of replication. The at least one selection marker may select cells comprising the vector of the present invention. In particular, it may be provide the cell with an advantage over cells not comprising the vector with the at least one selection marker. Examples of selection markers include antibiotic resistance selection markers, which confer a resistance to an otherwise toxic substance, e.g. beta-lactamase or hygromycin B phosphotransferase, which confers antibiotic resistance for eukaryotes and prokaryotes, or auxotrophic selection markers, that confer the ability to grow in a medium lacking an essential growth substance. Selection markers, also called selectable markers, may be specific for prokaryotic cells or eukaryotic cells or may be a selection marker for both, prokaryotic and eukaryotic cells. The at least one origin of replication may be a eukaryotic or prokaryotic origin of replication. Examples of an origin of replication include pSClOl, ColEl, pBR322 and pACYC177.

[0209] In some embodiments of the present invention, the vector is a viral vector, an artificial chromosome or a plasmid.

[0210] In a sixth aspect, the present invention relates to a eukaryotic cell comprising a nucleic acid of the first or third aspect, a protein of the second aspect, an expression construct of the fourth aspect or a vector of the fifth aspect.

[0211] All embodiments mentioned for the first, second, third, fourth and / or fifth aspect of the invention apply for the sixth aspect of the invention mutatis mutandis. In some embodiments, the nucleic acid, expression construct or vector of the present invention may be integrated into the genome of the eukaryotic cell or may be present as extrachromosomal DNA. The eukaryotic cell may comprise the nucleic acid, expression construct or vector of the present invention transiently.

[0212] In another embodiment of the present invention, the nucleic acid, expression construct or vector is integrated into the genome, i.e. a chromosome, of the eukaryotic cell. The integration may be specific or random. Exemplary modes of integration include transposons, such as sleeping beauty transposons or via a retrovirus.

[0213] In some embodiments of the present invention, the eukaryotic cell is an insect or mammalian cell. In some embodiments of the present invention, the eukaryotic cell is a mouse, rat, goat, enopus laevis or human cell. In some embodiments of the present invention, the eukaryotic cell is a human cell.

[0214] In some embodiments of the present invention, the eukaryotic cell is selected from the group consisting of HeLa cell, CHO cell, Sf21 cell, Pichia pastoris cell and HEK293 cell. In some embodiments of the present invention, the eukaryotic cell is a CHO or HEK293 cell. In some embodiments of the present invention, the eukaryotic cell is a HEK293 cell.

[0215] In a seventh aspect, the present invention relates to a method for the production of a protein comprising a truncated GFAP, in particular of the second aspect, comprising:

[0216] (a) providing a eukaryotic cell of the sixth aspect,

[0217] (b) culturing the eukaryotic cell in a cell culture medium under conditions that allow the cell to express the protein comprising a truncated GFAP encoded by a nucleic acid of the first or third aspect and thereby producing the protein comprising a truncated GFAP, and

[0218] (c) obtaining the protein comprising a truncated GFAP produced in step (b).

[0219] All embodiments mentioned for the first, second, third, fourth, fifth and / or sixth aspect of the invention apply for the seventh aspect of the invention and vice versa. The eukaryotic cell may be provided in step (a) by adding the cell to the cell culture medium. It may be added by ways known to the skilled person. For example, the cell may be added in the form of a suspension in a similar or different cell culture medium in which the cell grew before addition or by addition of a stock, such as a glycerol stock, to the cell culture medium.

[0220] Conditions that allow the cell to express the encoded protein may include the presence of inducer, in case the expression is controlled by an inducible promoter, or may include the culturing of the cells under conditions that allows the cell to express genes controlled by a constitutive promoter. In case of employing an inducer to control expression in step (b), the cell may be cultured to a desired cell density before addition of the inducer.

[0221] The protein comprising a truncated GFAP produced in step (b) may be obtained by any method known to the skilled person. In particular, it may be obtained by collection of the cell, with an optional subsequent lysation of the cell, or cell medium supernatant, optionally with a centrifugation and / or filtering step.

[0222] In some embodiments of the present invention, in step (a) the eukaryotic cell is provided by adding the cell to a cell culture medium.

[0223] In some embodiments of the present invention, the method further comprises

[0224] (d) purifying the protein comprising a truncated GFAP obtained in step (c).

[0225] The purification of step (d) may comprise any method known to the skilled person suitable for purifying a protein. In some embodiments of the present invention, the protein is purified via affinity chromatography, in particular immobilized metal affinity chromatography, from a cell medium supernatant or from a cell lysate. In some embodiments of the present invention, the cell is harvested and lysed before the purification in step (d). The methods of the present invention may comprise further purification steps, such as size exclusion chromatography.

[0226] In some embodiments of the present invention, the nucleic acid comprised in the eukaryotic cell codes for a protein comprising a truncated GFAP and at least one tag. In some embodiments of the present invention, the at least one tag is cleaved off the protein comprising a truncated GFAP during purification. In this embodiment, a linker sequence, said linker sequence comprising a protease cleavage site, is typically comprised between the at least one tag and the truncated GFAP.

[0227] In some embodiments of the present invention, the nucleic acid comprised in the eukaryotic cell codes for a protein comprising a truncated GFAP and a eukaryotic secretion tag. In this embodiment, the protein comprising a truncated GFAP and a eukaryotic secretion tag is secreted into the cell culture medium. In some embodiments of the present invention, the signal peptide of the eukaryotic secretion tag is cleaved off the protein comprising a truncated GFAP during secretion into the cell culture medium.

[0228] In some embodiments of the present invention, the protein comprising a truncated GFAP obtained in step (c) or (d) of a method of the present invention is in a further step (e) lyophilized. This lyophilized protein comprising a truncated GFAP may subsequently be reconstituted.

[0229] In an eighth aspect, the present invention relates to a protein comprising a truncated GFAP obtainable, in particular obtained, by a method of the seventh aspect.

[0230] All embodiments mentioned for the first, second, third, fourth, fifth, sixth and / or seventh aspect of the invention apply for the eighth aspect of the invention mutatis mutandis.

[0231] In a ninth aspect, the present invention relates to a composition comprising a protein of the second or eighth aspect.

[0232] All embodiments mentioned for the first, second, third, fourth, fifth, sixth, seventh and / or eighth aspect of the invention apply for the ninth aspect of the invention mutatis mutandis.

[0233] In some embodiments of the present invention, the composition is a lyophilisate or a solution.

[0234] In some embodiments of the present invention, the composition is an aqueous solution. In some embodiments of the present invention, the composition is a lyophilisate. A lyophilised composition, i.e. a lyophilisate, of the present invention has the advantage that it can be stored under more amicable conditions compared to, for example, a solution, because the protein is less prone to degradation by chemicals or hydrolisation. A solution as a composition may need storage at lower temperatures, such as 4 °C, but not too low, such as below 0 °C or freezing temperature, in order to prolong shelf-life, but not destroy the protein due to freezing. At the same time, a lyophilised composition can be stored in freezing conditions that other components of a kit with the lyophilisate might require, because no or only minimal amounts of water are present in the lyophilisate and therefore no damage due to freezing water occurs.

[0235] In some embodiments of the present invention, at least 70 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % of the protein comprising a truncated GFAP of the present invention in the composition is reconstitutable upon resolubilisation in deionized water.

[0236] In some embodiments, the percentage of reconstituted protein comprising a truncated GFAP is determined by

[0237] (A) preparing a solution comprising the protein comprising a truncated GFAP;

[0238] (B) lyophilising a part of the solution obtained in step (A);

[0239] (C) solubilizing the lyophilisate obtained in step (B) in deionized water;

[0240] (D) measuring the concentration of the protein comprising a truncated GFAP in the solutions obtained in step (A) and (C); and

[0241] (E) calculating the percentage of reconstituted protein comprising a truncated GFAP by comparing the concentration of the protein comprising a truncated GFAP in the solution obtained in step (A) with the concentration of the protein comprising a truncated GFAP in the solution obtained in step (C).

[0242] In some embodiments, the percentage of reconstituted protein comprising a truncated GFAP is determined by (A) preparing a solution comprising the protein comprising a truncated GFAP;

[0243] (B) lyophilising a part of the solution obtained in step (A);

[0244] (C) solubilizing the lyophilisate obtained in step (B) in deionized water;

[0245] (D) measuring the amount of the protein comprising a truncated GFAP in the solutions obtained in step (A) and (C);

[0246] (E) calculating the amount of the protein comprising a truncated GFAP in the lyophilisate obtained in step (B) with the amount of the protein comprising a truncated GFAP in the solution obtained in step (A) and the volume of the solution used in step (B); and

[0247] (F) calculating the percentage of reconstituted protein comprising a truncated GFAP by comparing the amount of the protein comprising a truncated GFAP in the lyophilisate obtained in step (B) with the amount of the protein comprising a truncated GFAP in the solution obtained in step (C).

[0248] In some embodiments of the present invention, the deionized water used to solubilize the lyophilisate in step (c) has a conductivity of smaller than 1 pS / cm at 25 °C. In some embodiments, the deionized water is Grade 2 water according to ISO 3696: 1987.

[0249] The concentration of the protein comprising GFAP can be measured in step (D) by methods known to the skilled person. In some embodiments of the present invention, in step (D) the concentration is measured via mass spectrometry or an immunoassay, in particular a sandwich immunoassay.

[0250] A specific example for a method for determining percentage of reconstituted protein is disclosed in the appended Example 3.

[0251] In some embodiments of the present invention, the solution used for solubilizing the lyophilisate in step (C) is an aqueous solution. Typically, a solution comprising a buffering agent, salt(s) and / or further ingredients, such as nonionic surfactant, is used. In some embodiments, the lyophilisate is solubilized in step (C) in a solution with the same ingredients of a solution that has been used in the preparation of the solution of step (A).

[0252] In a tenth aspect, the present invention relates to a kit comprising a protein of the second or eight aspect or at least one composition of the ninth aspect.

[0253] All embodiments mentioned for the first, second, third, fourth, fifth, sixth, seventh, eighth and / or ninth aspect of the invention apply for the tenth aspect of the invention mutatis mutandis.

[0254] In some embodiments of the present invention, the kit of the present invention comprises in separate container at least two compositions of the present invention with different concentrations of the protein of the present invention.

[0255] In some embodiments of the present invention, the kit is for use in the calibration of a measurement system for the in vitro quantification of GFAP or a variant thereof in a sample.

[0256] A kit of the present invention may be used for calibrating assays, such as immune assays. The calibration may be a new calibration with various increasing concentrations of the GFAP calibrator, or a one- or two-point calibration with measuring one or two concentrations of the calibrator. In a one- or two-point calibration, the measured concentration of the calibrator is used to calibrate the assay by correlating the measured concentration to a previously made master calibration curve.

[0257] In some embodiments of the present invention, the kit comprises a composition of the present invention in a container. Suitable container for a solid or liquid composition are known to the skilled person and may be of glass, optionally with a plastic cap, or plastic.

[0258] In some embodiments of the present invention, the kit comprises several containers each comprising a composition of the present invention with the same concentration of the protein of the present invention. In some embodiments of the present invention, the kit comprises a first container, a second container and optionally further containers, wherein each of the first, second and further containers comprises a composition of the present invention and the concentration of the protein of the present invention comprised in each composition of the first, second and optionally further container is different to the concentration of the protein comprising a truncated GFAP in each of the other containers. For example, the concentration of the protein comprising a truncated GFAP is different in the composition in the first container compared to the concentration of the protein comprising a truncated GFAP comprised in the composition in the second container.

[0259] In this embodiment, the kit may comprise more than one first, second and / or further container, wherein each first, second and further container comprises the same concentration of the protein of the present invention.

[0260] In an eleventh aspect, the present invention relates to a use of a protein of the second or eighth aspect, a composition of the ninth aspect or a kit of the tenth aspect for calibrating a measurement system for the in vitro quantification of GFAP or a variant thereof in a sample.

[0261] All embodiments mentioned for the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and / or tenth aspect of the invention apply for the eleventh aspect of the invention mutatis mutandis.

[0262] In some embodiments of the present invention, the sample is a biological fluid sample.

[0263] In some embodiments of the present invention, the biological fluid sample is whole blood, in particular capillary or venous blood, serum, blood plasma, saliva or cerebrospinal fluid. In some embodiments of the present invention, the biological fluid sample is whole blood. In some embodiments of the present invention, the biological fluid sample is serum. In some embodiments of the present invention, the biological fluid sample is blood plasma.

[0264] In a twelfth aspect, the present invention relates to a method for calibrating a measurement system for the in vitro quantification of GFAP or a variant thereof in a sample comprising a step of measuring at least once with the measurement system at least one composition according to the ninth aspect having a known concentration of the protein comprising a truncated GFAP.

[0265] All embodiments mentioned for the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth and / or eleventh aspect of the invention apply for the twelfth aspect of the invention mutatis mutandis.

[0266] In further embodiments, the present invention relates to the following items:

[0267] 1. A nucleic acid coding for a protein said protein comprising a truncated glial fibrillary acidic protein (GFAP), wherein the truncated GFAP comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 85 % sequence identity to SEQ ID NO: 2, wherein the protein does

[0268] (i) neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 10 consecutive amino acids, and / or

[0269] (ii) neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 10 consecutive amino acids.

[0270] 2. The nucleic acid of item 1, wherein the nucleic acid is DNA.

[0271] 3. The nucleic acid of item 1 or 2, wherein the amino acid sequence of the truncated GFAP is derived from a human GFAP.

[0272] 4. The nucleic acid of any one of items 1 to 3, wherein the protein does neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 10 consecutive amino acids, and neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 10 consecutive amino acids.

[0273] 5. The nucleic acid of any one of items 1 to 4, wherein the protein does not comprise a fragment of at least 9 consecutive amino acids of

[0274] (i) SEQ ID NO: 5 and / or

[0275] (ii) SEQ ID NO: 6.

[0276] 6. The nucleic acid of any one of items 1 to 4, wherein the protein does not comprise a fragment of at least 8 consecutive amino acids of (i) SEQ ID NO: 5 and / or

[0277] (ii) SEQ ID NO: 6.

[0278] 7. The nucleic acid of any one of items 1 to 4, wherein the protein does not comprise a fragment of at least 7 consecutive amino acids of

[0279] (i) SEQ ID NO: 5 and / or

[0280] (ii) SEQ ID NO: 6.

[0281] 8. The nucleic acid of any one of items 1 to 4, wherein the protein does not comprise a fragment of at least 6 consecutive amino acids of

[0282] (i) SEQ ID NO: 5 and / or

[0283] (ii) SEQ ID NO: 6.

[0284] 9. The nucleic acid of any one of items 1 to 4, wherein the protein does not comprise a fragment of at least 5 consecutive amino acids of

[0285] (i) SEQ ID NO: 5 and / or

[0286] (ii) SEQ ID NO: 6.

[0287] 10. The nucleic acid of any one of items 1 to 4, wherein the protein does not comprise a fragment of at least 4 consecutive amino acids of

[0288] (i) SEQ ID NO: 5 and / or

[0289] (ii) SEQ ID NO: 6.

[0290] 11. The nucleic acid of any one of items 1 to 4, wherein the protein does not comprise a fragment of at least 3 consecutive amino acids of

[0291] (i) SEQ ID NO: 5 and / or

[0292] (ii) SEQ ID NO: 6.

[0293] 12. The nucleic acid of any one of items 1 to 11, wherein the amino acid sequence of the truncated GFAP comprises or consists of SEQ ID NO: 3, SEQ ID NO: 4 or a variant thereof having at least 85 % sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4.

[0294] 13. The nucleic acid of any one of items 1 to 11, wherein the amino acid sequence of the truncated GFAP comprises or consists of SEQ ID NO: 3, SEQ ID NO: 4 or a variant thereof having at least 90 % sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4.

[0295] 14. The nucleic acid of any one of items 1 to 11, wherein the amino acid sequence of the truncated GFAP comprises or consists of SEQ ID NO: 3, SEQ ID NO: 4 or a variant thereof having at least 95 % sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4.

[0296] 15. The nucleic acid of any one of items 1 to 11, wherein the amino acid sequence of the truncated GFAP comprises or consists of SEQ ID NO: 3 or SEQ ID NO: 4.

[0297] 16. The nucleic acid of any one of items 1 to 11, wherein the truncated GFAP comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 90 % sequence identity to SEQ ID NO: 2.

[0298] 17. The nucleic acid of any one of items 1 to 11, wherein the truncated GFAP comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 95 % sequence identity to SEQ ID NO: 2.

[0299] 18. The nucleic acid of any one of items 1 to 11, wherein the truncated GFAP comprises the amino acid sequence of SEQ ID NO: 2.

[0300] 19. The nucleic acid of any one of items 1 to 11, wherein the amino acid sequence of the truncated GFAP consists of SEQ ID NO: 2 or a variant thereof having at least 85 % sequence identity to SEQ ID NO: 2.

[0301] 20. The nucleic acid of any one of items 1 to 11, wherein the amino acid sequence of the truncated GFAP consists of SEQ ID NO: 2 or a variant thereof having at least 90 % sequence identity to SEQ ID NO: 2.

[0302] 21. The nucleic acid of any one of items 1 to 11, wherein the amino acid sequence of the truncated GFAP consists of SEQ ID NO: 2 or a variant thereof having at least 95 % sequence identity to SEQ ID NO: 2.

[0303] 22. The nucleic acid of any one of items 1 to 11, wherein the amino acid sequence of the truncated GFAP consists of SEQ ID NO: 2. 23. The nucleic acid of any one of items 1 to 22, wherein the protein further comprises at least one tag.

[0304] 24. The nucleic acid of item 23, wherein at least one tag is a fluorescent tag, an affinity tag, solubility enhancing tag and / or eukaryotic secretion tag.

[0305] 25. The nucleic acid of item 23 or 24, wherein at least one tag is a His-tag, FLAG- tag, HA-tag, Avi-tag, strep-tag, MBP-tag, Fc-tag, GFP-tag or GST-tag.

[0306] 26. The nucleic acid of any one of items 23 to 25, wherein at least one tag is present at the N-terminus or C-terminus of the protein.

[0307] 27. The nucleic acid of any one of items 23 to 26, wherein a linker sequence is comprised between the at least one tag and the truncated GFAP.

[0308] 28. The nucleic acid of any one of items 23 to 27, wherein at least one linker sequence comprises a protease cleavage site.

[0309] 29. The nucleic acid of item 28, wherein the protease cleavage site is a TEV protease cleavage site, a furin cleavage site, IgA protease cleavage site, a SUMO protease cleavage site, a NEDD8 protease cleavage site, a ubiquitin protease cleavage site, a Thrombin cleavage site, a Factor Xa cleavage site or a HRV 3C protease cleavage site.

[0310] 30. The nucleic acid of any one of items 27 to 29, wherein at least one linker sequence comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and amino acid sequences having at least 85 % sequence identity to any one of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

[0311] 31. The nucleic acid of any one of items 27 to 29, wherein at least one linker sequence comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and amino acid sequences having at least 90 % sequence identity to any one of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. 32. The nucleic acid of any one of items 27 to 29, wherein at least one linker sequence comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and amino acid sequences having at least 95 % sequence identity to any one of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

[0312] 33. The nucleic acid of any one of items 27 to 29, wherein at least one linker sequence comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: H and SEQ ID NO: 12.

[0313] 34. The nucleic acid of any one of items 23 to 33, wherein the tag is a eukaryotic secretion tag.

[0314] 35. The nucleic acid of any one of items 23 to 34, wherein the eukaryotic secretion tag comprises a signal peptide from a preprotein of a eukaryotic secretory protein.

[0315] 36. The nucleic acid of item 35, wherein the eukaryotic secretion tag consists of a signal peptide from a preprotein of a eukaryotic secretory protein.

[0316] 37. The nucleic acid of any one of items 23 to 35, wherein the eukaryotic secretion tag is a preprotein of a eukaryotic secretory protein or part thereof, wherein the fragment comprises the signal peptide and at least one further amino acid.

[0317] 38. The nucleic acid of any one of items 23 to 37, wherein the eukaryotic secretion tag is selected from the group consisting of the albumin domain 1 (ADI domain), albumin and serotransferrin.

[0318] 39. The nucleic acid of any one of items 23 to 38, wherein the eukaryotic secretion tag is derived from a mammalian protein, in particular a human protein.

[0319] 40. The nucleic acid of any one of items 23 to 39, wherein the eukaryotic secretion tag comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and amino acid sequences having at least 85 % sequence identity to any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16. 41. The nucleic acid of any one of items 23 to 39, wherein the eukaryotic secretion tag comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and amino acid sequences having at least 90 % sequence identity to any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

[0320] 42. The nucleic acid of any one of items 23 to 39, wherein the eukaryotic secretion tag comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and amino acid sequences having at least 95 % sequence identity to any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

[0321] 43. The nucleic acid of any one of items 23 to 39, wherein the eukaryotic secretion tag comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

[0322] 44. The nucleic acid of any one of items 23 to 43, wherein the protein comprises the eukaryotic secretion tag at its N-terminus.

[0323] 45. The nucleic acid of any one of items 23 to 44, wherein the protein further comprises a eukaryotic secretion tag and a further tag.

[0324] 46. The nucleic acid of item 45, wherein at least one further tag is N-terminal or C- terminal of the truncated GFAP.

[0325] 47. The nucleic acid of any one of items 23 to 46, wherein the protein further comprises a eukaryotic secretion tag at its N-terminus and an affinity tag at its C- terminus.

[0326] 48. The nucleic acid of any one of items 23 to 47, wherein the protein further comprises a eukaryotic secretion tag at its N-terminus and a His-tag at its C-terminus.

[0327] 49. The nucleic acid of any one of items 23 to 48, wherein a linker sequence is comprised between the eukaryotic secretion tag and the truncated GFAP and optionally between the further tag and the truncated GFAP. 50. The nucleic acid of any one of items 23 to 49, wherein a linker sequence is comprised between the eukaryotic secretion tag and the truncated GFAP and between the further tag and the truncated GFAP, wherein the further tag is an affinity-tag, typically a His-tag.

[0328] 51. The nucleic acid of any one of items 1-11, 23-29, 34-39 or 44-50, wherein the protein comprises or consists of the amino acid sequence of SEQ ID NO: 18 or a variant thereof having at least 85 % sequence identity to SEQ ID NO: 18.

[0329] 52. The nucleic acid of any one of items 1-11, 23-29, 34-39 or 44-50, wherein the protein comprises or consists of the amino acid sequence of SEQ ID NO: 18 or a variant thereof having at least 90 % sequence identity to SEQ ID NO: 18.

[0330] 53. The nucleic acid of any one of items 1-11, 23-29, 34-39 or 44-50, wherein the protein comprises or consists of the amino acid sequence of SEQ ID NO: 18 or a variant thereof having at least 95 % sequence identity to SEQ ID NO: 18.

[0331] 54. The nucleic acid of any one of items 1-11, 23-29, 34-39 or 44-50, wherein the protein comprises or consists of the amino acid sequence of SEQ ID NO: 18.

[0332] 55. A protein comprising a truncated GFAP, wherein the truncated GFAP comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 85 % sequence identity to SEQ ID NO: 2, wherein the protein does

[0333] (i) neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 10 consecutive amino acids, and / or

[0334] (ii) neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 10 consecutive amino acids.

[0335] 56. The protein of item 55, wherein the protein is a recombinant protein.

[0336] 57. The protein of item 55 or 56, wherein the amino acid sequence of the truncated GFAP is derived from a human GFAP.

[0337] 58. The protein of any one of items 55 to 57, wherein the protein does neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 10 consecutive amino acids, and neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 10 consecutive amino acids.

[0338] 59. The protein of any one of items 55 to 58, wherein the protein does not comprise a fragment of at least 9 consecutive amino acids of

[0339] (i) SEQ ID NO: 5 and / or

[0340] (ii) SEQ ID NO: 6.

[0341] 60. The protein of any one of items 55 to 58„ wherein the protein does not comprise a fragment of at least 8 consecutive amino acids of

[0342] (i) SEQ ID NO: 5 and / or

[0343] (ii) SEQ ID NO: 6.

[0344] 61. The protein of any one of items 55 to 58„ wherein the protein does not comprise a fragment of at least 7 consecutive amino acids of

[0345] (i) SEQ ID NO: 5 and / or

[0346] (ii) SEQ ID NO: 6.

[0347] 62. The protein of any one of items 55 to 58„ wherein the protein does not comprise a fragment of at least 6 consecutive amino acids of

[0348] (i) SEQ ID NO: 5 and / or

[0349] (ii) SEQ ID NO: 6.

[0350] 63. The protein of any one of items 55 to 58„ wherein the protein does not comprise a fragment of at least 5 consecutive amino acids of

[0351] (i) SEQ ID NO: 5 and / or

[0352] (ii) SEQ ID NO: 6.

[0353] 64. The protein of any one of items 55 to 58„ wherein the protein does not comprise a fragment of at least 4 consecutive amino acids of

[0354] (i) SEQ ID NO: 5 and / or

[0355] (ii) SEQ ID NO: 6.

[0356] 65. The protein of any one of items 55 to 58„ wherein the protein does not comprise a fragment of at least 3 consecutive amino acids of

[0357] (i) SEQ ID NO: 5 and / or (ii) SEQ ID NO: 6.

[0358] 66. The protein of any one of items 55 to 65, wherein the amino acid sequence of the truncated GFAP comprises or consists of SEQ ID NO: 3, SEQ ID NO: 4 or a variant thereof having at least 85 % sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4.

[0359] 67. The protein of any one of items 55 to 65, wherein the amino acid sequence of the truncated GFAP comprises or consists of SEQ ID NO: 3, SEQ ID NO: 4 or a variant thereof having at least 90 % sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4.

[0360] 68. The protein of any one of items 55 to 65, wherein the amino acid sequence of the truncated GFAP comprises or consists of SEQ ID NO: 3, SEQ ID NO: 4 or a variant thereof having at least 95 % sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4.

[0361] 69. The protein of any one of items 55 to 65, wherein the amino acid sequence of the truncated GFAP comprises or consists of SEQ ID NO: 3 or SEQ ID NO: 4.

[0362] 70. The protein of any one of items 55 to 65, wherein the truncated GFAP comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 90 % sequence identity to SEQ ID NO: 2.

[0363] 71. The protein of any one of items 55 to 65, wherein the truncated GFAP comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 95 % sequence identity to SEQ ID NO: 2.

[0364] 72. The protein of any one of items 55 to 65, wherein the truncated GFAP comprises the amino acid sequence of SEQ ID NO: 2.

[0365] 73. The protein of any one of items 55 to 65, wherein the amino acid sequence of the truncated GFAP consists of SEQ ID NO: 2 or a variant thereof having at least 85 % sequence identity to SEQ ID NO: 2.

[0366] 74. The protein of any one of items 55 to 65, wherein the amino acid sequence of the truncated GFAP consists of SEQ ID NO: 2 or a variant thereof having at least 90 % sequence identity to SEQ ID NO: 2. 75. The protein of any one of items 55 to 65, wherein the amino acid sequence of the truncated GFAP consists of SEQ ID NO: 2 or a variant thereof having at least 95 % sequence identity to SEQ ID NO: 2.

[0367] 76. The protein of any one of items 55 to 65, wherein the amino acid sequence of the truncated GFAP consists of SEQ ID NO: 2.

[0368] 77. The protein of any one of items 55 to 76, wherein the protein further comprises at least a part of a eukaryotic secretory protein.

[0369] 78. The protein of item 77, wherein the eukaryotic secretory protein is a full length eukaryotic secretory protein.

[0370] 79. The protein of item 77 or 78, wherein the eukaryotic secretory protein is selected from the group consisting of the albumin domain 1 (ADI domain), albumin and serotransferrin.

[0371] 80. The protein of any one of items 77 to 79, wherein the eukaryotic secretory protein or part thereof is derived from a mammalian eukaryotic secretory protein, in particular a human eukaryotic secretory protein.

[0372] 81. The protein of any one of items 77 to 80, wherein the eukaryotic secretory protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and sequences having at least 85 % sequence identity to any one of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0373] 82. The protein of any one of items 77 to 80, wherein the eukaryotic secretory protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and sequences having at least 90 % sequence identity to any one of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0374] 83. The protein of any one of items 77 to 80, wherein the eukaryotic secretory protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and sequences having at least 95 % sequence identity to any one of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. 84. The protein of any one of items 77 to 80, wherein the eukaryotic secretory protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

[0375] 85. The protein of any one of items 77 to 84, wherein the protein comprises the eukaryotic secretory protein or part thereof at its N-terminus.

[0376] 86. The protein of any one of items 55 to 85, wherein the protein further comprises at least one tag.

[0377] 87. The protein of item 86, wherein at least one tag is a fluorescent tag, an affinity tag and / or solubility enhancing tag.

[0378] 88. The protein of item 86 or 87, wherein at least one tag is a His-tag, FLAG-tag, HA-tag, Avi-tag, strep-tag, MBP-tag, Fc-tag, GFP-tag or GST-tag.

[0379] 89. The protein of any one of items 86 to 88, wherein at least one tag is N-terminal or C-terminal of the truncated GFAP.

[0380] 90. The protein of any one of items 86 to 89, wherein at least one tag is present at the N-terminus or C-terminus of the protein.

[0381] 91. The protein of any one of items 77 to 90, wherein a linker sequence is comprised between the eukaryotic secretory protein or part thereof and the truncated GFAP, and / or at least one tag and the truncated GFAP.

[0382] 92. The protein of item 91, wherein at least one linker sequence comprises a protease cleavage site.

[0383] 93. The protein of item 92, wherein the protease cleavage site is a TEV protease cleavage site, a furin cleavage site, IgA protease cleavage site, a SUMO protease cleavage site, a NEDD8 protease cleavage site, a ubiquitin protease cleavage site, a Thrombin cleavage site, a Factor Xa cleavage site or a HRV 3C protease cleavage site.

[0384] 94. The protein of any one of items 91 to 93, wherein at least one linker sequence comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and amino acid sequences having at least 85 % sequence identity to any one of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

[0385] 95. The protein of any one of items 91 to 93, wherein at least one linker sequence comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and amino acid sequences having at least 90 % sequence identity to any one of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

[0386] 96. The protein of any one of items 91 to 93, wherein at least one linker sequence comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and amino acid sequences having at least 95 % sequence identity to any one of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

[0387] 97. The protein of any one of items 91 to 93, wherein at least one linker sequence comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: H and SEQ ID NO: 12.

[0388] 98. The protein of any one of items 77 to 97 further comprising at least a part of a eukaryotic secretory protein and a tag.

[0389] 99. The protein of any one of items 77 to 98 further comprising at least a part of a eukaryotic secretory protein at the N-terminus and an affinity tag at its C-terminus.

[0390] 100. The protein of any one of items 77 to 99 further comprising at least a part of a eukaryotic secretory protein at the N-terminus and a His-tag at its C-terminus.

[0391] 101. The protein of any one of items 77 to 100, wherein a linker sequence is comprised between the eukaryotic secretory protein or part thereof, and the truncated GFAP and between the tag and the truncated GFAP.

[0392] 102. The protein of any one of items 77 to 101, wherein a linker sequence is comprised between the eukaryotic secretory protein and the truncated GFAP, and between the tag and the truncated GFAP, wherein the tag is an affinity-tag, typically a His-tag.

[0393] 103. The protein of any one of items 77 to 102, wherein the protein comprising a truncated GFAP has a polydispersity index of at most 1.1, typically at most 1.05, more typically at most 1.01 when in solution.

[0394] 104. The protein of any one of items 55-65, 77-80, 85-93 or 98-103, wherein the protein comprises or consists of the amino acid sequence of SEQ ID NO: 17 or a variant thereof having at least 85 % sequence identity to SEQ ID NO: 17.

[0395] 105. The protein of any one of items 55-65, 77-80, 85-93 or 98-103, wherein the protein comprises or consists of the amino acid sequence of SEQ ID NO: 17 or a variant thereof having at least 90 % sequence identity to SEQ ID NO: 17.

[0396] 106. The protein of any one of items 55-65, 77-80, 85-93 or 98-103, wherein the protein comprises or consists of the amino acid sequence of SEQ ID NO: 17 or a variant thereof having at least 95 % sequence identity to SEQ ID NO: 17.

[0397] 107. The protein of any one of items 55-65, 77-80, 85-93 or 98-103, wherein the protein comprises or consists of the amino acid sequence of SEQ ID NO: 17.

[0398] 108. A nucleic acid coding for a protein of any one of items 55 to 107.

[0399] 109. An expression construct comprising a nucleic acid of any one of items 1-54 or 108.

[0400] 110. The expression construct of item 109 comprising a promoter, a terminator, a 5' UTR and / or a 3' UTR.

[0401] 111. The expression construct of item 110, wherein the promoter is an inducible promoter.

[0402] 112. The expression construct of item 110, wherein the promoter is a constitutive promoter. 113. The expression construct of item 109 or 110, wherein the expression construct comprises the human cytomegalovirus immediate-early enhancer / promoter region.

[0403] 114. The expression construct of any one of items 109 to 113, wherein the expression construct comprises the bovine growth hormone polyadenylation signal.

[0404] 115. A vector comprising an expression construct of any one of items 109 to 114.

[0405] 116. The vector of item 115 comprising at least one selection marker and / or at least one origin of replication.

[0406] 117. The vector of item 115 or 116, wherein the vector is a viral vector, artificial chromosome or a plasmid.

[0407] 118. A eukaryotic cell comprising a nucleic acid of any one of items 1-54 or 108, a protein of any one of items 55 to 107, an expression construct of any one of items 109 to 114 or a vector of any one of items 115 to 117.

[0408] 119. The eukaryotic cell of item 118, wherein the eukaryotic cell is a human cell.

[0409] 120. The eukaryotic cell of item 118 or 119, wherein the eukaryotic cell is a CHO or HEK293 cell.

[0410] 121. A method for the production of a protein comprising a truncated GFAP, in particular of items 55 to 107, comprising:

[0411] (a) providing a eukaryotic cell of any one of items 118 to 120,

[0412] (b) culturing the eukaryotic cell in a cell culture medium under conditions that allow the cell to express the protein comprising a truncated GFAP encoded by a nucleic acid of any one of items 1 to 54 or 108 and thereby producing the protein comprising a truncated GFAP, and

[0413] (c) obtaining the protein comprising a truncated GFAP produced in step (b).

[0414] 122. The method of item 121 comprising further

[0415] (d) purifying the protein comprising a truncated GFAP obtained in step (c). 123. The method of item 122, wherein the protein is purified via affinity chromatography, in particular immobilized metal affinity chromatography, from a cell medium supernatant or from a cell lysate.

[0416] 124. The method of any one of items 121 to 123, wherein the nucleic acid comprised in the eukaryotic cell codes for a protein comprising a truncated GFAP and at least one tag.

[0417] 125. The method of item 124, wherein the at least one tag is cleaved off the protein comprising a truncated GFAP during purification.

[0418] 126. The method of any one of items 121 to 125, wherein the nucleic acid comprised in the eukaryotic cell codes for a protein comprising a truncated GFAP and a eukaryotic secretion tag.

[0419] 127. The method of item 126, wherein the signal peptide of the eukaryotic secretion tag is cleaved off the protein comprising a truncated GFAP during secretion into the cell culture medium.

[0420] 128. The method of any one of items 121 to 127, wherein the protein comprising a truncated GFAP obtained in step (c) or (d) of a method of the present invention is in a further step (e) lyophilized.

[0421] 129. A protein comprising a truncated GFAP obtainable, in particular obtained, by a method of any one of items 121 to 128.

[0422] 130. A composition comprising a protein of any one of items 55-107 or 129.

[0423] 131. The composition of item 130, wherein the composition is a lyophilisate or a solution, typically a lyophilisate.

[0424] 132. The composition of item 130 or 131, wherein the composition is a lyophilisate and at least 70 % of the protein comprising a truncated GFAP of any one of items 55 to 107 or item 129 in the composition is reconstitutable upon resolubilisation in deionized water. 133. The composition of item 130 or 131, wherein the composition is a lyophilisate and at least 90 % of the protein comprising a truncated GFAP of any one of items 55 to 107 or item 129 in the composition is reconstitutable upon resolubilisation in deionized water.

[0425] 134. The composition of item 130 or 131, wherein the composition is a lyophilisate and at least 95 % of the protein comprising a truncated GFAP of any one of items 55 to 107 or item 129 in the composition is reconstitutable upon resolubilisation in deionized water.

[0426] 135. The composition of item 130 or 131, wherein the composition is a lyophilisate and at least 97 % of the protein comprising a truncated GFAP of any one of items 55 to 107 or item 129 in the composition is reconstitutable upon resolubilisation in deionized water.

[0427] 136. The composition of item 130 or 131, wherein the composition is a lyophilisate and at least 98 % of the protein comprising a truncated GFAP of any one of 55 to 107 or item 129 in the composition is reconstitutable upon resolubilisation in deionized water.

[0428] 137. The composition of item 130 or 131, wherein the composition is a lyophilisate and at least 99 % of the protein comprising a truncated GFAP of any one of items 55 to 107 or item 129 in the composition is reconstitutable upon resolubilisation in deionized water.

[0429] 138. The composition of any one of items 132 to 137, wherein the percentage of reconstituted protein comprising a truncated GFAP is determined by

[0430] (A) preparing a solution comprising the protein comprising a truncated GFAP;

[0431] (B) lyophilising a part of the solution obtained in step (A);

[0432] (C) solubilizing the lyophilisate obtained in step (B) in deionized water;

[0433] (D) measuring the concentration of the protein comprising a truncated GFAP in the solutions obtained in step (A) and (C);

[0434] (E) calculating the percentage of reconstituted protein comprising a truncated GFAP by comparing the concentration of the protein comprising a truncated GFAP in the solution obtained in step (A) with the concentration of the protein comprising a truncated GFAP in the solution obtained in step (C).

[0435] 139. The composition of item 138, wherein in step (D) the concentration is measured via mass spectrometry or an immunoassay, in particular a sandwich immunoassay.

[0436] 140. A kit comprising a protein of any one of items 55-107 or 129, or at least one composition of any one of items 130 to 139.

[0437] 141. The kit of item 140 comprising a first container, a second container and optionally further containers, wherein each of the first, second and optionally further containers comprises a composition of any one of items 130 to 139 and the concentration of the protein of any one of items 55-107 or 129 comprised in each composition of the first, second and optionally further container is different to the concentration of the protein comprising a truncated GFAP in each of the other containers.

[0438] 142. The kit of item 140 or 141 for use in the calibration of a measurement system for the in vitro quantification of GFAP or a variant thereof in a sample.

[0439] 143. Use of a protein of any one of items 55-107 or 129, a composition of any one of items 130 to 139 or a kit of any one of items 140 to 142 for calibrating a measurement system for the in vitro quantification of GFAP or a variant thereof in a sample.

[0440] 144. The use of item 143, wherein the sample is a biological fluid sample.

[0441] 145. The use of item 144, wherein the biological fluid sample is whole blood, in particular capillary or venous blood, serum, blood plasma, saliva or cerebrospinal fluid.

[0442] 146. A method for calibrating a measurement system for the in vitro quantification of GFAP or a variant thereof in a sample comprising a step of measuring at least once with the measurement system at least one composition according to any one of items 130 to 139 having a known concentration of the protein comprising a truncated GFAP. SEQ SEQ SEQ

[0443] ID Name Amino Acid Sequence

[0444] NO SEQ

[0445] ID Name Amino Acid Sequence

[0446] NO SEQ

[0447] ID Name Amino Acid Sequence

[0448] NO SEQ SEQ

[0449] ID Name Amino Acid Sequence

[0450] NO SEQ

[0451] ID Name Amino Acid Sequence

[0452] NO SEQ

[0453] ID Name Amino Acid Sequence

[0454] NO

[0455] Examples

[0456] The following examples are provided to illustrate, but not to limit the presently claimed invention.

[0457] Example 1

[0458] Fragments of GFAP, in particular truncated GFAP, were tested for secretory expression. Truncated GFAP was expressed as albumin fusion protein in order to secret it to the cell culture supernatant.

[0459] For transient gene expression, the FreeStyle™ 293-F expression system (Thermo Fischer Scientific) was used. The following expression constructs were designed, each featuring the same human cytomegalovirus immediate-early enhancer / promoter region, and the coding gene regions being followed by the same bovine growth hormone polyadenylation signal:

[0460] • GFAP-His8, GFAP HUMAN, Uniprot Q14136 residues 1-432, plus C- terminal His-Tag

[0461] • ALB(Albumin)-GFAP-His8, ALBU HUMAN, Uniprot P02768 residues 1- 609, fusion with GFAP_HUMAN, Uniprot Q14136 residues 1-432, plus C- terminal His-Tag

[0462] • ALB-GFAP AA 69-377-His8, ALBU HUMAN, Uniprot P02768 residues 1-609, fusion with GFAP_HUMAN, Uniprot Q14136 residues 69-377, plus C- terminal His-Tag

[0463] Full-length human glial fibrillary acidic protein, GFAP -Hiss, is a cytosolic, aggregation prone protein: Cultures at ~ 2 * 10E6 cells / ml were transfected with the expression plasmid at a concentration of 0.5 mg / 1 cell culture complexed by the PEIpro™ transfection reagent (Polyplus) according to the manufacturer's guidelines. 72 hrs post-transfection, the cell pellet was collected by centrifugation and stored at -80 °C for cell lysis and subsequent protein purification. The frozen cells were thawed, resuspended in 50 mM HEPES pH 7.5, 300 mM NaCl, 5 mM MgCh, 5 % glycerol, 0.1 % polysorbate 20. Protease inhibitor (10 tablets Complete EDTA free (Roche) per 100 ml) and DNAse I were added to the buffer directly before use. A Potter-Elvehjem Homogenizer was used for cell lysis. For purification, the clarified lysate was applied on IMAC chromatography. The His-tagged GFAP was eluted with imidazole and dialyzed into the final storage buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 6.5 % Sucrose).

[0464] In contrast, C-terminal fusion to human albumin resulted in secretion of both ALB- GFAP-Hiss and ALB-GFAP AA 69-377-Hiss into the HEK293 culture supernatant, facilitating the purification: 7 days post-transfection, the cell supernatant was collected by centrifugation and stored at -80 °C for subsequent protein purification by IMAC chromatography. The His-tagged ALB-GFAP fusion proteins were eluted with imidazole and polished by preparative size exclusion chromatography using a Superose™ 200 column. The purified ALB-GFAP -Hiss did not show any signs of glycosylation when analysed by mass spectrometry.

[0465] Example 2

[0466] The purified GFAP constructs were characterized by SDS-PAGE under reducing conditions using Coomassie blue staining for protein detection (a), SEC-MALS HPLC (b) and nDSF / DLS temperature ramp experiment (c).

[0467] (a) SDS-PAGE

[0468] For SDS-PAGE analysis, approx. 5 pg protein treated with NuP AGE™ SDS Sample Buffer (4x) (Invitrogen) with 100 mM DTT were treated according to the manufacturer’s instructions and applied on a NuPAGE™ 4-12% Bis-Tris Gel (Invitrogen). The staining was performed with Roti® Blue Quick (Roth).

[0469] Both proteins exhibit in the SDS-PAGE an apparent molecular weight of about 100 kDa and a good purity (see FIG. 1 A & B).

[0470] (b) SEC-MALS HPLC

[0471] The SEC-MALS data were collected using a Superdex® 200 increase 10 / 300 GL column (GE Healthcare, Freiburg, Germany) connected to a HPLC system (Ultimate 3000 RS, Thermo Fisher Scientific GmbH, Dreieich, Germany), a multiangel lightscattering detector (miniDAWN® TREOS® II, Wyatt Technology, Santa Barbara, United States) and a refractive index detector (Optilab® T.rEX, Wyatt Technology, Santa Barbara, United States).

[0472] A running buffer of 50 mM potassium dihydrogen phosphate, 100 rnM potassium chloride, 1 mM EDTA, 0.01 % Na-Azid, pH 7.5 was used at a flow rate of 0.7 mL / min. For SEC -MALS, an injection volume was chosen to inject approx. 50-100 pg protein sample.

[0473] Data were analysed using ASTRA software (version 7.3.2.21; Wyatt Technology, Santa Barbara, United States).

[0474] SEC: Size Exclusion Chromatography, MALS: Multi angle static light scattering, HPLC: high-performance liquid chromatography.

[0475] Table 1 : Calculated molecular weight and polydispersity index

[0476] Mw: weight average molecular weight, Mn: number average molecular weight, kDa: 1 kDa = 1000 g / mol

[0477] The main peak of ALB-GFAP-Hiss in the chromatogram covers molecular weights from 300 kDa to 2000 kDa (see FIG. 2). The molecular weight of ALB-GFAP AA 69-377-Hiss in the chromatogram is stable distributed around 400 kDa, which indicates a homogeneous tetramer (see FIG. 3).

[0478] (c) nDSF / DLS temperature ramp experiment

[0479] Thermal Unfolding analysis was performed using the Prometheus Panta system and Panta Analysis Software (NanoTemper Technologies GmbH, Miinchen, Germany). Sample handling was performed with Prometheus NT.48 Series nanoDSF Grade Standard Capillaries. The samples with a protein concentration of approx. 1 mg / ml were scanned with a temperature ramp of 1.5 K / min from 20 °C to 95 °C.

[0480] Data were analyzed using Prometheus Panta Analyzing Software (NanoTemper Technologies GmbH, Munich, Germany).

[0481] With nDSF data of the various GFAP Albumin fusion, no defined temperature induced transition of the GFAP domain could be observed.

[0482] The hydrodynamic radii measured with DLS showed a significant higher thermostability of ALB-GFAP AA 69-377-Hiss compared to the full length ALB- GFAP-Hiss (see FIG. 4).

[0483] Example 3

[0484] The various GFAP fusion protein stock solutions were diluted in Elecsys® Diluent Universal, catalogue number 03183971122 (Roche Diagnostics GmbH, Germany) to give a calibration curve from 0 - 25 ng / ml. Two different recombinant GFAP fusion protein preparations were assessed for recovery after lyophilization. GFAP (Uniprot: P14136) and a fragment covering amino acids 69-377 where expressed as albumin fusion proteins. 0.5 ml aliquots were frozen at -80 °C in 3 ml glass vials and subsequently lyophilized with a Christ Beta 2-16 according to the following protocol. The freeze dryer was turned on 15 min before placing the glass vials with the frozen aliquots into the freeze drying chamber. The lyophilisation was performed for 1 day at not more than 100 mbar absolute pressure. After the lyophilisation, the glass vials were removed from the chamber and closed.

[0485] The lyophilized aliquots were reconstituted with deionized water for 15 or 30 min and assessed for signal recovery.

[0486] Elecsys® Assay:

[0487] Antibodies against GFAP were generated at Roche Diagnostics GmbH (Penzberg, Germany), Immunoglobulin G was purified by Protein A chromatography (MabSelect SuRe, GE Healthcare, Uppsala Sweden) according to the supplier’s manual. A sandwich immunoassay was developed on the Elecsys® platform. The electrochemiluminescence technology on the Elecsys® platform is based on streptavidin-coated microbeads. Capture antibodies were biotinylated and detection antibodies labelled with ruthenium for signal generation as described previously (Hermanson GT. Bioconjugate techniques: Academic press; 2013). 30 pl of sample volume were assayed in a total reaction volume of 120 pl (30 pl sample, 33 pl reagent 1 with capture antibodies, 33 pl reagent 2 with detection antibodies and 24 pl magnetic beads) with an incubation time of 18 min at 37 °C. The experiments were run on an Elecsys® e411 or e601 system. Table 2:

[0488] Surprisingly the fragment (GFAP AA 69-377) was stable upon lyophilisation and showed a signal recovery of 97 - 105 % (see Table 2). The full length showed a signal loss with a recovery of 7 - 17 %. The fragment showed a superior stability vs the full length protein.

Claims

Patent Claims1. A nucleic acid coding for a protein said protein comprising a truncated glial fibrillary acidic protein (GFAP), wherein the truncated GFAP comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 85 % sequence identity to SEQ ID NO: 2, wherein the protein does(i) neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 10 consecutive amino acids, and / or(ii) neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 10 consecutive amino acids.

2. The nucleic acid of claim 1, wherein the protein does neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 10 consecutive amino acids, and neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 10 consecutive amino acids.

3. A protein comprising a truncated GFAP, wherein the truncated GFAP comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 85 % sequence identity to SEQ ID NO: 2, wherein the protein does(i) neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 10 consecutive amino acids, and / or(ii) neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 10 consecutive amino acids.

4. The protein of claim 3, wherein the protein does neither comprise the amino acid sequence of SEQ ID NO: 5 nor fragments thereof of at least 10 consecutive amino acids, and neither comprise the amino acid sequence of SEQ ID NO: 6 nor fragments thereof of at least 10 consecutive amino acids.

5. The protein of claim 3 or 4, wherein the protein comprising a truncated GFAP has a polydispersity index of at most 1.1 when in solution.

6. An expression construct comprising a nucleic acid of claim 1 or 2.

7. A vector comprising an expression construct of claim 6.

8. A eukaryotic cell comprising a nucleic acid of claim 1 or 2, a protein of any one of claims 3 to 5, an expression construct of claim 6 or a vector of claim 7.

9. A method for the production of a protein comprising a truncated GFAP comprising:(a) providing a eukaryotic cell of claim 8,(b) culturing the eukaryotic cell in a cell culture medium under conditions that allow the cell to express the protein comprising a truncated GFAP encoded by a nucleic acid of claim 1 or 2 and thereby producing the protein comprising a truncated GFAP, and(c) obtaining the protein comprising a truncated GFAP produced in step (b).

10. The method of claim 9 comprising further(d) purifying the protein comprising a truncated GFAP obtained in step (c).

11. A protein comprising a truncated GFAP obtainable, in particular obtained, by a method of claim 9 or 10.

12. A composition comprising a protein of any one of claims 3 to 5 or 11.

13. The composition of claim 12, wherein the composition is a lyophilisate and at least 70 % of the protein comprising a truncated GFAP of any one of claims 3 to 5 or 11 in the composition is reconstitutable upon resolubilisation in deionized water.

14. A kit comprising a protein of any one of claims 3 to 5 or 11, or at least one composition of claim 12 or 13.

15. Use of a protein of any one of claims 3 to 5 or 11, a composition of claim 12 or 13, or a kit of claim 14 for calibrating a measurement system for the in vitro quantification of GFAP or a variant thereof in a sample.

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