Expression of glial fibrillary acidic protein

Abstract

The present invention relates to: nucleic acids encoding a protein comprising a glial fibrillary acidic protein (GFAP) and a eukaryotic secretion tag; proteins comprising a GFAP and a eukaryotic secretion tag; expression constructs comprising a nucleic acid encoding a protein comprising a GFAP and a eukaryotic secretion tag; vectors comprising a nucleic acid or expression construct disclosed herein; and eukaryotic cells comprising a nucleic acid, expression construct, or vector disclosed herein. The present invention also relates to methods for producing a protein disclosed herein. The present invention also relates to compositions comprising a protein disclosed herein and kits comprising a protein and / or a composition disclosed herein. Furthermore, the present invention relates to the use of a protein, composition, and / or kit disclosed herein for calibrating a measurement system for the quantification of GFAP or a variant thereof in a sample. In vitro quantification of GFAP or a variant thereof in a sample.

Description

Technical Field

[0001] This invention relates to: nucleic acids encoding proteins comprising glial fibrillary acidic protein (GFAP) and eukaryotic secretion tags; proteins comprising GFAP and eukaryotic secretion tags; expression constructs comprising nucleic acids encoding proteins comprising GFAP and eukaryotic secretion tags; vectors comprising the nucleic acids or expression constructs disclosed herein; and eukaryotic cells comprising the nucleic acids, expression constructs, or vectors disclosed herein. The invention also relates to methods for generating the proteins disclosed herein. Furthermore, the invention relates to compositions comprising the proteins disclosed herein and kits comprising the proteins and / or compositions disclosed herein. Additionally, the invention relates to the proteins, compositions, and / or kits disclosed herein for calibrating GFAP or its variants in samples. in vitro Applications of quantitative measurement systems. Background Technology

[0002] 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 main intermediate filament expressed in mature astrocytes.

[0003] GFAP is used as a blood-derived biomarker for the diagnosis and assessment of traumatic brain or spinal cord injuries, as well as other conditions affecting the central nervous system, such as inflammatory diseases like multiple sclerosis or cancers like gliomas. For example, the FDA approved a clinical blood test for assessing mild traumatic brain injury in 2018.

[0004] For protein quantification using different assays (e.g., immunoassays, immunosandwich assays, or ELISA), assay calibration is crucial. For this purpose, so-called calibrators are used. A calibrator is a protein standard used at a known concentration to determine the concentration of a target protein in a sample via interpolation. There are certain requirements for using calibrators. One such requirement is that they must be stable so that they can be stored for extended periods, such as as lyophilized products. Unstable proteins cannot be used as calibrators because the detectable amount of the protein changes significantly after short-term storage, thus defeating the purpose of using a calibrator at a known concentration.

[0005] To date, the only available source of GFAP calibrators is human brain tissue. This presents several challenges: supply depends on the availability of sufficient human brain tissue, which must also be disease-free. Furthermore, GFAP is purified from human brain tissue using urea, which denatures GFAP to maintain its solubility. However, urea can interfere with subsequent uses in the assay, thus requiring careful purification.

[0006] Therefore, it is of particular interest to obtain recombinant GFAPs with the advantages of being safer and more readily available, as well as more stable GFAPs for use as calibrators.

[0007] To date, only prokaryotic expression systems have been attempted to express GFAP. CN113005134A discloses a method for expressing GFAP in... E. coli The expression of codon-optimized GFAP is shown in the figure. E. coli The full-length GFAP expressed in the medium cannot fold correctly and forms inclusion bodies containing GFAP during expression. Purifying GFAP from inclusion bodies requires further steps, including denaturation of the inclusion bodies and the protein therein, and protein refolding, which carries the risk of incorrect refolding. Furthermore, the yield is much lower, and multiple invasive chemicals must be used. CN113005134A also discloses fusing prokaryotic secretion signals to GFAP. However, purification is not performed from cell culture medium but by lysing cells, meaning secretion is unsuccessful and the protein cannot be expressed in a soluble manner.

[0008] Quinlan et al. (Quinlan) et al. The paper "Expression in Escherichia coli of fragments of glial fibrillary acidic protein: characterization, assembly properties and paracrystal formation," in the *Journal of Cell Science*, 93, 71-83, 1989, discloses multiple fragments of mouse GFAP in... E. coli The expression in the text. However, the fragments only exist in inclusion bodies and must be painstakingly purified and refolded.

[0009] Chen and Liem (Chen and Liem, The endless story of the glial fibrillaryacidic protein, Journal of Cell Science, 107, 2299-2311, 1994) published the expression of GFAP deletion mutants with partial deletions at the N or C ends in human cancer cells to investigate the role of different parts of GFAP in intermediate filament assembly. They found that only a portion of the C-terminal tail domain of GFAP could be deleted without interfering with self-assembly, while the intact N-terminal head domain was essential for self-assembly. They did not purify or analyze the storage stability of these mutants.

[0010] Therefore, there remains a need to produce GFAPs using safer, easier, and more reliable methods, particularly for their use as calibrators, enabling those skilled in the art to obtain stable GFAPs. Furthermore, cost reduction and / or ensuring long-term product availability are also beneficial. Summary of the Invention

[0011] In a first aspect, the present invention relates to nucleic acids encoding proteins, the proteins comprising

[0012] (i) Glial fibrous acidic protein (GFAP), wherein the GFAP comprises at least 250 consecutive amino acids of the amino acid sequence of SEQ ID NO: 1 or a variant of the amino acid sequence having at least 90% sequence identity with it, and

[0013] (ii) Eukaryotic secretion tags.

[0014] The inventors were surprised to discover that GFAP and GFAP fragments can be recombinantly expressed in a soluble form using a eukaryotic secretion tag that leads to the secretion of GFAP. This soluble form of secretory expression avoids any need for protein refolding. The nucleic acids disclosed herein are particularly suitable for the secretory expression of proteins containing GFAP.

[0015] The methods and techniques provided herein enable those skilled in the art to produce GFAPs that are safer (because they are derived from disease-free sources), easier (because they do not require protein refolding), and more reliable (because they are recombinant expression proteins rather than isolated from natural sources, and therefore do not depend on the availability of natural sources).

[0016] Because of the eukaryotic secretion tag, when expressed in eukaryotic cells, the protein containing GFAP is secreted into the supernatant. This facilitates the production of GFAP in a more convenient way, for example, as a calibrator, since cells do not have to lyse and production can be carried out continuously.

[0017] As can be seen from the appended embodiments, the GFAP obtained by the method of the present invention exhibits good stability when attacked in stability tests.

[0018] The inventors were surprised to discover that it is possible to secrete normal intracellular GFAP using eukaryotic cells, and therefore even soluble GFAP can be obtained. Furthermore, the advantage of secretion using eukaryotic cells is that insoluble GFAP can be easily separated from soluble, correctly folded GFAP without the need for cell lysis and protein refolding. As can be seen from the accompanying examples, when the eukaryotic secretion tag is recombined and linked to a truncated GFAP, the truncated version of GFAP can also be expressed and secreted.

[0019] In a second aspect, the present invention relates to a protein comprising: (i) a GFAP, wherein the GFAP comprises at least 250 consecutive amino acids of an amino acid sequence from SEQ ID NO: 1 or a variant of the amino acid sequence having at least 90% sequence identity with it, and (ii) At least a portion of eukaryotic secretory proteins.

[0020] The proteins in the second aspect, particularly the portions containing eukaryotic secretory proteins, typically lack any signal peptides from the pre-eukaryotic proteins because these signal peptides are cleaved during secretion. A specific example of a portion of a eukaryotic secretory protein is albumin domain 1, such as the albumin domain 1 in SEQ ID NO: 7.

[0021] In a fourth aspect, the present invention relates to expression constructs comprising nucleic acids of the first or third aspect.

[0022] In a fifth aspect, the present invention relates to a carrier comprising the expression construct of the fourth aspect.

[0023] In a sixth aspect, the present invention relates to eukaryotic cells comprising the nucleic acid of the first aspect, the expression construct of the fourth aspect, or the vector of the fifth aspect.

[0024] In a seventh aspect, the present invention relates to a method for producing a protein comprising GFAP, comprising: (a) Provides eukaryotic cells for the sixth aspect, (b) Culture eukaryotic cells in cell culture medium under the following conditions: allowing cells to express and secrete a protein containing GFAP encoded by a nucleic acid of the first or third aspect into the cell culture medium; and thereby producing a protein containing GFAP, and (c) Obtain the protein containing GFAP secreted in step (b).

[0025] In an eighth aspect, the present invention relates to proteins comprising GFAP, which can be obtained, in particular, by the method described in the seventh aspect.

[0026] In a ninth aspect, the present invention relates to a composition comprising the protein of the second or eighth aspect.

[0027] In a tenth aspect, the present invention relates to a kit comprising: a protein of the second or eighth aspect, or at least a composition of the ninth aspect.

[0028] In the eleventh aspect, the present invention relates to proteins of the second or eighth aspect, compositions of the ninth aspect, or kits of the tenth aspect for calibrating GFAP or its variants in samples. in vitro Applications of quantitative measurement systems.

[0029] In a twelfth aspect, the present invention relates to a method for calibrating GFAP or its variants in a sample. in vitro A method for quantitative measurement system, comprising the step of: using the measurement system to perform at least one measurement on a composition containing at least one GFAP protein according to the ninth aspect and having a known concentration. Attached Figure Description

[0030] Figure 1 shows the SDS-PAGE analysis results of insulin signal peptide GFAP FLAG tag (1), albumin domain 1 linker GFAP FLAG tag (2), albumin linker GFAP FLAG tag (3), albumin domain 1 linker 2 GFAP FLAG tag (4), and serum transferrin linker GFAP FLAG tag (5) under reducing conditions.

[0031] Figures 2A and 2B show the SDS-PAGE analyses of albumin (ALB)-GFAP-His8 (A) and ALB-GFAP_69-377-His8 (B) under reducing conditions: (A) Line M: Marker SeeBlue ™Plus2 pre-stained (Invitrogen), line 1: ALB-GFAP-His8 (B) Line M: Marker SeeBlue ™ Plus2 pre-staining (Invitrogen), Line 1: High molecular weight impurities separated in the final purification step, Line 2: ALB-GFAP_AA 69-377-His8, Line 3: Low molecular weight impurities separated in the final purification step. Detailed Implementation

[0032] Before describing the invention in detail below, it should be understood that the invention is not limited to the specific embodiments and examples described herein, as these embodiments and examples can vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which will be limited only by the appended claims. Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0033] This specification incorporates numerous references throughout. Each of these references (including all patents, patent applications, scientific publications, manufacturer's instructions, user manuals, etc.), whether cited above or below, is incorporated herein by reference in its entirety. In the event of any conflict between the definitions or teachings of such incorporated references and those cited in this specification, the definitions and teachings in this specification shall prevail.

[0034] Elements of the invention will now be described. These elements are listed along with specific embodiments; however, it should be understood that they can be combined in any manner and in any number to create other embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention solely to the explicitly described embodiments. This description should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, unless the context otherwise requires, any arrangement and combination of the elements described herein should be considered as disclosed in the specification of this application.

[0035] The following definitions and embodiments apply in their entirety to this disclosure, and are particularly applicable to all aspects and embodiments of the invention.

[0036] As used in this specification and the appended claims, unless otherwise expressly stated, the singular forms “a,” “an,” “the,” and “the” include the plural referents.

[0037] The word "comprise" and its variations such as "comprises" and "comprising" should be understood to imply the inclusion of the stated integers or steps or groups of integers or steps, but not to exclude any other integers or steps or groups of integers or steps. In some embodiments, the word "comprise" may include "consisting of".

[0038] The use of alternatives (e.g., "or") should be understood to mean any one, both, or any combination of alternatives.

[0039] The term “and / or” should be understood to mean either or both of the alternatives.

[0040] Percentages, concentrations, amounts, and other numerical data may be expressed or presented in the format of "range" throughout this document. It should be understood that this range format is used solely for convenience and brevity, and therefore should be flexibly interpreted to include not only the values ​​explicitly listed as range limits, but also all individual values ​​or subranges covered by the range, as if each value and subrange were explicitly listed. For example, the numerical range "4% to 20%" should be interpreted to include not only the explicitly listed values ​​of 4% to 20%, but also the individual values ​​and subranges within the indicated range. Therefore, this numerical range includes individual values ​​such as 4, 5, 6, 7, 8, 9, 10, ... 18, 19, 20% and subranges such as 4-10%, 5-15%, 10-20%, etc. The same principle applies to ranges referencing minimum or maximum values. Furthermore, this interpretation should apply regardless of the breadth or characteristics of the range being described.

[0041] As used herein and unless otherwise stated, the term “about” should be understood to be used synonymously with the term “approximately”. Demonstratively and unless otherwise stated, the use of the term “about” when combined with a specified numerical value or range indicates a value or range slightly greater than or slightly less than the specified value, within ±15% of the specified value, within ±10% of the specified value, within ±5% of the specified value, or conveniently within ±2% of the specified value. Therefore, such values ​​are covered by the scope of the claims that reference the terms “about” or “approximately”.

[0042] As used in this disclosure, "% w / v" refers to weight-volume percentage, which is a unit of concentration expressed as a percentage of the amount of solute measured in grams (g) as a percentage of the total volume of the solution in milliliters (ml).

[0043] As used in this disclosure, "% v / v" refers to volume percentage, which is a unit of concentration expressed as a percentage of the total volume of the solution (in milliliters (ml)) of a particular solute.

[0044] The terms "lyophilising" or "lyophilization" refer to the process of freezing a substance by freezing it and then reducing the ambient pressure (e.g., below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or lower), causing the freezing medium in the substance to sublimate directly from the solid phase to the gas phase. Therefore, the terms "lyophilizing" and "lyophilization" are used interchangeably herein.

[0045] The term “reconstruction” involves adding a solvent (such as water) to a dried product (such as a lyophilized product) to restore it to a liquid state (such as its original liquid state).

[0046] In the context of this disclosure, the term "recombinant" means "prepared by genetic engineering." In some embodiments, the "recombinant protein" in the context of this disclosure is not naturally occurring.

[0047] As used herein, the term "truncated" refers to a non-naturally occurring protein that differs from the naturally occurring protein from which it is derived in that at least one amino acid is missing from one or both ends of the N-terminus or C-terminus of the protein. Deletions in the protein sequence may not be considered truncated. Such truncations can be generated through recombination or by using peptidases. For example, the deletion of amino acids 1 to 20 at the N-terminus of a 100-amino acid-long protein results in a truncated protein lacking amino acids 1 to 20.

[0048] As used herein, the terms “room temperature” and “ambient temperature” are used interchangeably and refer to temperatures ranging from at least about 15°C, for example 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.

[0049] The term "cryoprotectant" refers to substances added to a formulation to protect the active ingredient during freezing.

[0050] The term "lyophilization protectant" refers to substances added to a formulation to protect the active ingredient during freeze-drying.

[0051] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, peptides or nucleic acids that exist in organisms (including viruses), can be isolated from natural sources, and have not been intentionally modified in a laboratory are naturally occurring. The term "found in nature" means "existing in nature," including known objects as well as objects that have not yet been discovered and / or are isolated from nature but may be discovered in the future and / or are isolated from natural sources. The term "natural" refers to naturally occurring objects.

[0052] According to the present invention, the term "peptide" refers to a molecule comprising about two or more, about three or more, about four or more, about six or more, about eight or more, about ten or more, about thirteen or more, about sixteen or more, about 20 or more, and up to about 50, about 100, or about 150 amino acids linked together by peptide bonds. The term "polypeptide" refers to a large peptide, particularly a peptide having at least about 151 amino acids. Both "peptide" and "polypeptide" are protein molecules. Therefore, the terms "peptide," "protein," and "polypeptide" are generally used as synonyms herein.

[0053] According to the present invention, a protein "lacking amino acids 1 to 68 corresponding to SEQ ID NO: 1" refers to a protein that neither contains nor contains any part of amino acids 1 to 68 of SEQ ID NO: 1. The portion of amino acids 1 to 68 of SEQ ID NO: 1 can 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. Preferably, it is a portion with a shorter minimum length. According to the present invention, a protein "lacking amino acids 378 to 432 corresponding to SEQ ID NO: 1" refers to a protein that neither contains nor contains any part of amino acids 378 to 432 of SEQ ID NO: 1. The portion of amino acids 378 to 432 of SEQ ID NO: 1 can 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. A shorter minimum length portion is preferred. For example, the sequence SAARRSYVSSGEM corresponding to amino acids 8 to 20 of SEQ ID NO: 1 is part of the amino acid sequence of SEQ ID NO: 1 and can be deleted from the protein according to the invention.

[0054] According to the present invention, a protein that is "a fragment that neither contains the amino acid sequence of SEQ ID NO: 5 nor contains at least 10 consecutive amino acids of SEQ ID NO: 5" or "a fragment that neither contains the amino acid sequence of SEQ ID NO: 6 nor contains at least 10 consecutive amino acids of SEQ ID NO: 6" refers to a protein in which, when sequence alignment is performed, all amino acids corresponding to the amino acids in the referenced SEQ ID sequence are missing (in their respective consecutive sequences); in terms of a fragment, it means that, when sequence alignment is performed with a protein, the amino acids of that fragment in their respective consecutive sequences are missing. A fragment can 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 reference sequence. In the present invention, fragments with a shorter minimum length are preferred. For example, the sequence SAARRSYVSSGEM corresponding to amino acids 8 to 20 of SEQ ID NO: 5 is a fragment of more than 10 consecutive amino acids missing from the protein according to the invention, which contains neither the amino acid sequence of SEQ ID NO: 5 nor a fragment containing at least 10 consecutive amino acids thereof.

[0055] For the purposes of this invention, a “variant” of an amino acid sequence (peptide or polypeptide) may include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term “variant” includes all mutants, splicing variants, post-translational modification variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those that are naturally occurring. The term “variant” includes, in particular, fragments of an amino acid sequence. An amino acid insertion variant includes the insertion of one or more amino acids into a specific amino acid sequence. In the case of a variant with an inserted amino acid sequence, one or more amino acid residues are inserted into a specific site in the amino acid sequence, although random insertion and appropriate screening of the resulting product are also possible. An amino acid addition variant includes the fusion of one or more amino acid terms at the amino and / or carboxyl ends, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. An amino acid deletion variant is characterized by the removal of one or more amino acids from the sequence, such as the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Deletions can occur at any location in a protein. Amino acid deletion variants containing deletions at the N-terminus and / or C-terminus of a protein are also called N-terminal and / or C-terminal truncation variants. Amino acid substitution variants are characterized by the removal of at least one residue from the sequence and the insertion of another residue in its position. Modifications at non-conserved positions in the amino acid sequence between homologous peptides or polypeptides and / or substitution of amino acids with other amino acids having similar properties are preferred. In some embodiments, amino acid changes in peptide and polypeptide variants are conserved amino acid changes, i.e., substitutions of similar charged or uncharged amino acids. Conserved amino acid changes involve substitutions of one of the relevant amino acid families in their side chains. Naturally occurring amino acids are generally classified into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and non-polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. In some embodiments, conserved amino acid substitutions include substitutions within the group consisting of: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0056] "Sequence similarity" refers to the percentage of identical or conserved amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of identical amino acids between the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of identical nucleotides between the sequences. In some embodiments, the degree of similarity or identity of the entire length of a reference amino acid sequence is given. Alignments used to determine sequence similarity, such as sequence identity, can be performed using tools known in the art, such as optimal sequence alignment, for example using Align, with standard settings, preferably EMBOSS::Needle, matrix: Blosum62, vacancy open 10.0, vacancy extension 0.5.

[0057] The terms “%identical” and “%compatibility” or similar terms are intended to specifically refer to the percentage of identical nucleotides or amino acids in the best alignment between sequences to be compared. This percentage is purely statistical, and the differences between two sequences may, but are not necessarily, randomly distributed across the entire length of the sequences being compared. Comparison of two sequences is typically performed by comparing the sequences relative to fragments or a “comparison window” after best alignment in order to identify local regions of the corresponding sequences. The best alignment for comparison can be performed manually or with the aid of algorithms, such as Smith and Waterman's local homology algorithm, 1981, Ads App. Math. 2, 482; Needleman and Wunsch's local homology algorithm, 1970, J. Mol. Biol. 48, 443; Pearson and Lipman's similarity search algorithm, 1988, Proc. Natl Acad. Sci. USA 88, 2444; or with the aid of computer programs that use the aforementioned algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). The percentage identity of two sequences can be determined using the BLASTN or BLASTP algorithm (as available on the National Center for Biotechnology Information (NCBI) website). The algorithm parameters for the BLASTN algorithm on the NCBI website may include: (i) expected threshold set to 10; (ii) word size set to 28; (iii) maximum match within the query range set to 0; (iv) match / no-match score set to 1, -2; (v) gap cost set to linear; and (vi) filter for the low-complexity region being used. The algorithm parameters for the BLASTP algorithm on the NCBI website may include: (i) expected threshold set to 10; (ii) word size set to 3; (iii) maximum match within the query range set to 0; (iv) matrix set to BLOSUM62; (v) gap cost set to exist: 11, expansion: 1; and (vi) conditional combination score matrix adjustment.

[0058] Percentage identity is obtained by determining the number of identical positions corresponding to the sequences to be compared, dividing that number by the number of positions being compared (e.g., the number of positions in the reference sequence), and then multiplying the result by 100.

[0059] Throughout this disclosure, preferred references are those having higher sequence identity with a particular sequence (such as a sequence with a particular SEQ ID NO).

[0060] According to this disclosure, the homologous amino acid sequences exhibit at least 40%, particularly at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and for example at least 95%, at least 98%, or at least 99% identity of the amino acid residues.

[0061] The amino acid sequence variants described herein can be readily prepared by those skilled in the art, for example, through recombinant DNA manipulation. Operations for preparing DNA sequences of peptides or polypeptides with substitutions, additions, insertions, or deletions are described in detail in Molecular Cloning: e.g., A Laboratory Manual, 4th Edition, MR Green and J. Sambrook et al. (1989), eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012. Furthermore, the peptides, polypeptides, and amino acid variants described herein can be readily prepared using known peptide synthesis techniques, such as, for example, solid-phase synthesis and similar methods. A fragment or variant of an amino acid sequence (peptide or polypeptide) can be a “functional fragment” or a “functional variant.” The term “functional fragment” or “functional variant” of an amino acid sequence refers to any fragment or variant exhibiting one or more functional properties that are identical or similar to those functional properties of the amino acid sequence from which it originates; that is, it is functionally equivalent. Regarding the sequence of a binder (such as an antibody), a specific function is one or more binding activities exhibited by the amino acid sequence from which the fragment or variant originates. As used herein, the terms "functional fragment" or "functional variant" specifically refer to a variant molecule or sequence that comprises an amino acid sequence altered by one or more amino acids compared to the amino acid sequence of a parent molecule or sequence, and that still performs one or more functions of the parent molecule or sequence, such as binding to a target molecule. Modifications to 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 a functional fragment or functional variant may be reduced, but still significantly present; for example, the function of a functional fragment or functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence. However, the function of a functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0062] According to various embodiments of this disclosure, nucleic acids encoding peptides or polypeptides are taken up or introduced into cells, i.e., transfected or transduced into cells, which can then... In vitroThe peptide or polypeptide may be present in the subject, thereby leading to its expression. Cells may, for example, express the encoded peptide or polypeptide within the cell (e.g., in the cytoplasm and / or in the nucleus), secrete the encoded peptide or polypeptide, and / or express it on their surface. According to this disclosure, terms such as “expressing nucleic acid” and “encoding nucleic acid” or similar terms are used interchangeably herein, and with respect to a particular peptide or polypeptide, refers to the nucleic acid, if present in a suitable environment, such as within a cell, which can be expressed to produce the peptide or polypeptide.

[0063] The term “expression” as used in this article includes transcription and / or translation of a specific nucleotide sequence.

[0064] In the context of this disclosure, the term "transcription" refers to the process in which the genetic code in a DNA sequence is transcribed into RNA (specifically mRNA). Subsequently, the RNA can be translated into peptides or polypeptides.

[0065] Regarding RNA, the terms "expression" or "translation" refer to a process within the cell's ribosomes through which the mRNA chain directs the assembly of amino acid sequences to produce peptides or polypeptides.

[0066] The term "polydispersion index" ( M The polydispersity index is a measure of the inhomogeneity of molecular or particle size in a mixture. A collection of objects with uniform size, shape, or mass is called homogeneous. A sample of objects with inconsistent size, shape, and mass distributions is called inhomogeneous. The polydispersity index can be calculated using the formula... M = M w / M n Calculation, where M w It is the weight-average molecular weight, and M n It is the number-average molecular weight.

[0067] The polydispersity index can be calculated using methods known to those skilled in the art, particularly based on dynamic light scattering measurements performed through so-called cumulative analysis, as mentioned in the definition of "mean diameter." Under certain prerequisites, the polydispersity index can serve as a measure of the size distribution of an assemblages of nanoparticles.

[0068] The "hydrodynamic radius" of a particle (sometimes called the "Stokes radius" or "Stokes-Einstein radius") is the radius (complex hydrodynamic radius) of a hypothetical hard sphere diffusing at the same rate as the particle. The hydrodynamic radius relates to the particle's mobility, taking into account not only size but also solvent effects. For example, a smaller charged particle with stronger hydration may have a larger hydrodynamic radius than a larger charged particle with weaker hydration. This is because the smaller particle drags more water molecules as it moves through the solution. Since the actual size of particles in a solvent cannot be directly measured, the hydrodynamic radius can be defined by the Stokes-Einstein equations:

[0069] in k B Boltzmann's constant; T For temperature; η The viscosity of the solvent; and D The diffusion coefficient is denoted by . The diffusion coefficient can be determined experimentally, for example, by using dynamic light scattering (DLS). Therefore, one method for determining the hydrodynamic radius of a particle or particle group (such as the hydrodynamic radius of particles contained in the sample or control compositions disclosed herein, or the hydrodynamic radius of a particle peak obtained by subjecting the sample or control compositions to field flow fractionation) is to measure the DLS signal of the particle or particle group (such as the DLS signal of particles contained in the sample or control compositions disclosed herein, or the DLS signal of a particle peak obtained by subjecting the sample or control compositions to field flow fractionation).

[0070] As used in this article, "light scattering" refers to the physical process by which light is forced to deviate from one or more straight paths due to local inhomogeneities in the medium through which it passes.

[0071] The term "UV" refers to ultraviolet light and specifies the band of wavelengths in the electromagnetic spectrum from 10 nm to 400 nm, which is shorter than visible light but longer than X-rays.

[0072] The term "multi-angle light scattering" or "MALS" as used herein refers to a technique for measuring light scattered from a sample at multiple angles. In this respect, "multi-angle" means that scattered light can be detected at different discrete angles, for example, by means of a single detector that moves within a range including selected specific angles or by an array of detectors fixed at specific angular positions. In some embodiments, the light source used in MALS is a laser source (MALS: Multi-Angle Laser Light Scattering). Based on the MALS signal of the composition containing particles, and by using an appropriate form (e.g., Zimm plot, Berry plot, or Debye plot), the radius of gyration (Rg) can be determined, thereby determining the size of the particles (see, for example, Buchholz et al. (Electrophoresis 22 (2001), 4118-4128); BHZimm (J. Chem. Phys. 13 (1945), 141; P. Debye (J. Appl. Phys. 15 (1944): 338; and W. Burchard (Anal. Chem. 75 (2003), 4279-4291).

[0073] The term "dynamic light scattering" or "DLS" used in this paper refers to a technique for determining the size and size distribution spectrum of particles, particularly regarding the hydrodynamic radius of the particles. A monochromatic light source (typically a laser) is incident on the sample through a polarizer. The scattered light then passes through a second polarizer, where it is detected and the resulting image is projected onto a screen. Particles in the solution are struck by the light and diffract it in all directions. The diffracted light from the particles can interfere constructively (bright areas) or destructively (dark areas). This process is repeated over short time intervals, and the resulting speckle pattern set is analyzed by an autocorrelator, which compares the light intensity at each point over time.

[0074] As used herein, "static light scattering" or "SLS" refers to a technique for determining the size and size distribution spectrum of particles, particularly with respect to the radius of gyration and / or molar mass of the particles. High-intensity monochromatic light, typically a laser, is emitted from a solution containing the particles. One or more detectors are used to measure the scattering intensity at one or more angles. Angle dependence is essential for obtaining accurate measurements of the molar mass and size of macromolecules of all radii. Therefore, simultaneous measurements at multiple angles relative to the direction of incident light, termed multi-angle light scattering (MALS) or multi-angle laser light scattering (MALLS), are generally considered the standard implementation of static light scattering.

[0075] The term "C-terminus" refers to the carboxyl terminus of a peptide chain. It is also known as the "carboxyl terminus" or "COOH terminus." A peptide chain contains amino acids linked together by peptide bonds, which involve the C-terminus of the amino acids. α The amino and carboxyl groups at the C-terminus form an amino acid chain linked by the amino group of amino acid X+1 and the carboxyl group of amino acid X. Since the amino acids at both ends are not further linked to another amino acid, this leaves the amino group at one end and the carboxyl group at the other end free. The C-terminus is considered the end of the amino acid chain, and the N-terminus is considered the start of the chain. Therefore, the amino acid sequence starts from C... α Starting with an amino acid with a free amino group, up to C... α The amino acid with a free carboxyl group ends at this point.

[0076] The term "N-terminus" refers to the amino terminus of a peptide chain. It is also known as the "amino terminus," "amine terminus," or "NH2 terminus." The N-terminus is considered the starting point of the amino acid chain.

[0077] The term "calibrator" refers to a protein used as a protein standard, at a known concentration, for determining the concentration of a target protein in a sample by interpolation and / or for recalibrating a measurement system. After establishing a standard curve using calibrators of different concentrations, the measurement system can be recalibrated using a combination of one or two calibrators containing known concentrations. By measuring the composition at known concentrations, those skilled in the art can correlate the output signal from the measurement system with a specific concentration, and if a master standard curve has been previously calculated for the measurement system, a single-point recalibration is typically sufficient to reliably quantify the analyte in the sample across the entire range of the previously established master standard curve.

[0078] The term "tag" refers to an amino acid sequence that is recombinantly fused with a protein. Tags can be used for various purposes, such as protein purification or... In situ Protein detection. Some tags can even be used for two different purposes, such as the maltose-binding protein tag, which can be used as an affinity tag for both solubilization and purification. Tags can be fused to the N-terminus or C-terminus of a peptide or protein, or they can be located within the amino acid sequence. Proteins can have more than one tag, such as two affinity tags, which are used for two different purification steps or for different purposes, such as detection and purification. The terms “tag,” “protein tag,” and “peptide tag” are used as synonyms in this document. Examples of protein tags include His tags, FLAG tags, HA (hemagglutinin) tags, Avi tags, strep tags, MBP (maltose-binding protein) tags, Fc tags, GFP (green fluorescent protein) tags, or GST (glutathione S-transferase) tags.

[0079] An Fc tag is a protein tag containing at least a portion of a crystallizable fragment (Fc) of immunoglobulin G (IgG). It can be recombinantly fused to a peptide sequence, and the fused protein can subsequently be purified by protein A or protein G affinity chromatography. The affinity of protein A and protein G depends on the species and class of IgG from which the Fc tag originates.

[0080] The term "eukaryotic secretion tag" refers to a tag that can recombine with the amino acid sequence of a protein and cause the protein to be secreted from a eukaryotic cell. Eukaryotic secretion tags typically contain a signal peptide derived from a eukaryotic preprotein, but may also contain other parts of the preprotein, such as other amino acids that are not part of the signal peptide or other protein domains. In some embodiments, the eukaryotic secretion tag may also contain the complete amino acid sequence of the preprotein.

[0081] "Eukaryotic secretory proteins" are proteins secreted by eukaryotic cells. Eukaryotic secretory proteins are secreted by eukaryotic cells. Eukaryotic cell secretion typically involves the cleavage of a signal peptide during translocation into the endoplasmic reticulum. Therefore, eukaryotic secretory proteins lack a signal peptide. Furthermore, eukaryotic secretory proteins can have other post-translational modifications, such as cleavage of other parts of the protein. The term "eukaryotic secretory protein" specifically refers to the processed protein, not the preprotein or preprotoprotein.

[0082] "Preprotein" refers to a protein precursor, particularly a precursor of a secretory protein, which contains a signal peptide, also known as a presequence. Preproteins are secreted from the cell. In eukaryotic cells, the signal peptide is typically cleaved during or after the translocation of the preprotein's polypeptide chain to the endoplasmic reticulum. With the cleavage of the signal peptide, the preprotein is no longer present. The protein is then further processed in the ER and optionally in the Golgi apparatus, and ultimately delivered to the cell membrane for secretion.

[0083] The term "signal peptide" refers to an amino acid sequence of 14 to 35 amino acids, typically located at the N-terminus of a eukaryotic proprotein secreted by the cell. The core of the signal peptide is usually a hydrophobic amino acid segment, typically 5 to 16 amino acids long. This hydrophobic segment tends to form an α-helix. Approaching the C-terminus of the signal peptide, following the hydrophobic segment and preceding the amino acid sequence of the (pre)protein, is usually the cleavage site of a signal peptidase. The signal peptide typically acts as a recognition signal for a signal recognition particle (SPR), which usually binds to the signal peptide as it emerges from the ribosome during translation, pausing further translation, and is subsequently bound to an SRP receptor on the endoplasmic reticulum (ER). The nascent protein chain then translocates into the ER during translation. During or after translocation, the signal peptide is cleaved from the (pre)protein by a signal peptidase.

[0084] The term "fusion protein" refers to a polypeptide or protein comprising two or more subunits. Preferably, a fusion protein is a translational fusion between two or more subunits. Translational fusions can be generated by genetically engineering the coding nucleotide sequence of one subunit with the coding nucleotide sequence of another subunit within the reading frame. The subunits can be intercalated by adapters.

[0085] The term "expression construct" refers to a nucleic acid that contains a coding sequence and other nucleotide sequences operatively linked to that coding sequence, which are required for the expression of that coding sequence. While promoters and terminators are necessary for the efficient initiation and termination of transcription of the coding sequence into mRNA, other sequences such as 5' or 3' UTRs or enhancers are optional but contribute to the expression of the coding sequence.

[0086] The term "isolated" refers to a protein or polypeptide, nucleic acid, cell, or other designated 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. Isolated proteins or polypeptides, nucleic acids, cells, or other designated materials or components can often be in a non-natural form. For example, isolated nucleic acids can be nucleic acids that have been isolated from the cells in which they originally existed.

[0087] The amount, concentration, or level of protein in a composition (such as a solution) can be measured using various methods known to those skilled in the art. Such methods may include mass spectrometry, ultraviolet absorption spectrometry, Bradford assay, Lowry assay, or immunoassays such as ELISA. The percentage of a specific reconstituted protein from the lyophilized product can be determined by measuring the amount or concentration of protein in the solution obtained through reconstitution and comparing that amount or concentration with the amount or concentration of protein in the solution used to obtain the lyophilized product. When using concentration, the solution used to obtain the lyophilized product and the solution obtained through reconstitution must have the same volume or the concentration value must be adjusted for the same volume. For example, lyophilized product X is prepared using a 500 µL solution containing 2 mg / mL protein Y. The lyophilized product is reconstituted to 1 mL, and the concentration of protein Y in the reconstitution solution is 0.5 mg / mL. The adjusted concentration in the reconstitution solution is 1 mg / mL, and the percentage of reconstituted protein is 50% = (1 mg / mL ÷ 2 mg / mL) × 100.

[0088] Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0089] In a first aspect, the present invention relates to nucleic acids encoding proteins, the proteins comprising

[0090] (i) Glial fibrous acidic protein (GFAP), wherein the GFAP comprises at least 250 consecutive amino acids of the amino acid sequence of SEQ ID NO: 1 or a variant of the amino acid sequence having at least 90% sequence identity with it, and

[0091] (ii) Eukaryotic secretion tags.

[0092] In some embodiments of the present invention, the nucleic acid is DNA or RNA, particularly mRNA.

[0093] In some specific embodiments of the present invention, nucleic acid is DNA.

[0094] In some embodiments of the present invention, the nucleic acid is an isolated nucleic acid.

[0095] In some embodiments of the present invention, the amino acid sequence of GFAP is derived from human GFAP. In some embodiments of the present invention, the amino acid sequence of GFAP is derived from human GFAP identified by UniProt accession number P14136.

[0096] In some embodiments of the present invention, the GFAP comprises at least 260, at least 270, at least 280, at least 290, or at least 300 consecutive amino acids of the amino acid sequence of SEQ ID NO: 1 or a variant of the same amino acid sequence having at least 90% sequence identity. In some embodiments of the present invention, the GFAP comprises at least 300 consecutive amino acids of the amino acid sequence of SEQ ID NO: 1 or a variant of the same amino acid sequence having at least 90% sequence identity. In some embodiments of the present invention, the GFAP comprises at least 309 consecutive amino acids of the amino acid sequence of SEQ ID NO: 1 or a variant of the same amino acid sequence having at least 90% sequence identity.

[0097] In some embodiments of the present invention, the GFAP comprises at least 260, at least 270, at least 280, at least 290, or at least 300 consecutive amino acids of the amino acid sequence of SEQ ID NO: 1 or a variant of the same amino acid sequence having at least 95% sequence identity. In some embodiments of the present invention, the GFAP comprises at least 300 consecutive amino acids of the amino acid sequence of SEQ ID NO: 1 or a variant of the same amino acid sequence having at least 95% sequence identity. In some embodiments of the present invention, the GFAP comprises at least 309 consecutive amino acids of the amino acid sequence of SEQ ID NO: 1 or a variant of the same amino acid sequence having at least 95% sequence identity.

[0098] In some embodiments of the present invention, the GFAP comprises at least 260, at least 270, at least 280, at least 290, or at least 300 consecutive amino acids from the amino acid sequence of SEQ ID NO: 1. In some embodiments of the present invention, the GFAP comprises at least 300 consecutive amino acids from the amino acid sequence of SEQ ID NO: 1. In some embodiments of the present invention, the GFAP comprises at least 309 consecutive amino acids from the amino acid sequence of SEQ ID NO: 1.

[0099] In some embodiments of the invention, the protein is missing amino acids 1 to 68 corresponding to SEQ ID NO: 1 and / or amino acids 378 to 432 corresponding to SEQ ID NO: 1. Truncation of GFAP can lead to further increases in stability, solubility, and / or yield.

[0100] In some embodiments of the present invention, the protein is missing amino acids 1 to 68 corresponding to SEQ ID NO: 1 and amino acids 378 to 432 corresponding to SEQ ID NO: 1.

[0101] In some embodiments of the present invention, the protein contains neither the amino acid sequence of SEQ ID NO: 5 nor a fragment of at least 10 consecutive amino acids.

[0102] In some embodiments of the present invention, the protein contains neither the amino acid sequence of SEQ ID NO: 6 nor a fragment of at least 10 consecutive amino acids.

[0103] In some embodiments of the present invention, the protein contains neither the amino acid sequence of SEQ ID NO: 5 nor a fragment of at least 5 consecutive amino acids.

[0104] In some embodiments of the present invention, the protein contains neither the amino acid sequence of SEQ ID NO: 6 nor a fragment of at least 5 consecutive amino acids.

[0105] In some embodiments of the present invention, the protein contains neither the amino acid sequence of SEQ ID NO: 5 nor a fragment of at least three consecutive amino acids.

[0106] In some embodiments of the present invention, the protein contains neither the amino acid sequence of SEQ ID NO: 6 nor a fragment of at least three consecutive amino acids.

[0107] In some embodiments of the present invention, proteins

[0108] (i) It does not contain the amino acid sequence of SEQ ID NO: 5, nor does it contain a fragment of at least 10 consecutive amino acids therein, and / or

[0109] (ii) It does not contain the amino acid sequence of SEQ ID NO: 6, nor does it contain a fragment of at least 10 consecutive amino acids.

[0110] In some embodiments of the present invention, the protein does not contain 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 the following sequence: (i) SEQ ID NO: 5 and / or (ii) SEQ ID NO: 6.

[0111] In some embodiments of the present invention, GFAP comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof, the variant having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1.

[0112] In some embodiments of the present invention, GFAP comprises the amino acid sequence of SEQ ID NO: 1.

[0113] In some embodiments of the present invention, the amino acid sequence of GFAP consists of SEQ ID NO: 1 or a variant thereof, which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1.

[0114] In some embodiments of the present invention, the amino acid sequence of GFAP consists of SEQ ID NO: 1.

[0115] In some embodiments of the present invention, the amino acid sequence of GFAP consists of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or a variant thereof, the variant having at least 85%, at least 90% or at least 95% sequence identity with SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0116] In some embodiments of the present invention, the amino acid sequence of GFAP consists of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

[0117] In some embodiments of the present invention, the amino acid sequence of GFAP consists of SEQ ID NO: 2 or a variant thereof, which has at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 2.

[0118] In some embodiments of the present invention, the amino acid sequence of GFAP consists of SEQ ID NO: 2.

[0119] In some embodiments of the present invention, the eukaryotic secretion tag comprises a signal peptide derived from a preprotein of a eukaryotic secretory protein.

[0120] In some embodiments of the invention, the eukaryotic secretory tag is a preprotein or a portion of a eukaryotic secretory protein, wherein the portion comprises a signal peptide and at least one additional amino acid of the preprotein. In some embodiments of the invention, the portion is a domain of a eukaryotic secretory protein, i.e., a protein domain. In some embodiments of the invention, the domain is a functional domain of a eukaryotic secretory protein, performing the function normally assumed by that domain in the context of a full-length eukaryotic secretory protein.

[0121] In some embodiments of the present invention, the eukaryotic secretion tag comprises an insulin signaling peptide, an albumin domain 1 (AD1 domain), albumin, or serum transferrin. In some embodiments of the present invention, the eukaryotic secretion tag is selected from the group consisting of: insulin signaling peptide, albumin domain 1 (AD1 domain), albumin, and serum transferrin.

[0122] Eukaryotic secretion tags may offer additional advantages beyond secretion into cell culture media. Depending on the type of eukaryotic secretion tag, it may also increase solubility and / or be available for further purification after secretion. One such tag is albumin, which may contribute to further increased solubility.

[0123] In some embodiments of the present invention, the eukaryotic secretory tag is derived from mammalian proteins. In some embodiments of the present invention, the eukaryotic secretory tag is derived from mice, rats, goats, or African clawed frogs (Xenopus laevis). enopus laevis (or human protein.) Typically, eukaryotic secretory tags are derived from human proteins.

[0124] In some embodiments of the invention, the eukaryotic secretion tag comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

[0125] In some embodiments of the invention, the eukaryotic secretion tag comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

[0126] 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 an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

[0127] 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 and SEQ ID NO: 16.

[0128] In some embodiments of the invention, the protein includes a eukaryotic secretion tag at its N-terminus. It is preferable to fuse the eukaryotic secretion tag to the N-terminus of the protein because signal recognition particles, typically responsible for guiding ribosomes and nascent polypeptide chains to the ER, bind to the signal peptide at the N-terminus of the eukaryotic secretory protein. Depending on the signal peptide, the eukaryotic secretion tag may be contained at the N-terminus of the protein.

[0129] In some embodiments of the invention, the protein further comprises at least one additional tag. In addition to the eukaryotic secretion tag, the protein of the invention may also comprise additional tags that can increase solubility, such as an MBP (maltose-binding protein) tag, and / or can be used as a further purification step in affinity chromatography, such as an MBP tag or a His tag.

[0130] In some embodiments of the invention, at least one additional label is a fluorescent label, an affinity label, and / or a solubility-enhancing label.

[0131] In some embodiments of the present invention, at least one additional tag is a His tag, a FLAG tag, a HA tag, an Avi tag, a strep tag, a MBP tag, an Fc tag, a GFP tag, or a GST tag.

[0132] In some embodiments of the invention, at least one additional tag is located at the N-terminus or C-terminus of the GFAP.

[0133] In some embodiments of the invention, at least one additional tag is located at the C-terminus of the protein.

[0134] In some embodiments of the invention, the protein contains a eukaryotic secretion tag at its N-terminus and an affinity tag at its C-terminus.

[0135] In some embodiments of the invention, the protein contains a eukaryotic secretion tag at its N-terminus and a His tag at its C-terminus.

[0136] In some embodiments of the present invention, an adapter sequence is included between the eukaryotic secretory tag and the GFAP.

[0137] In some embodiments of the invention, a connector sequence is included between the additional tag and the GFAP.

[0138] In some embodiments of the invention, adapter sequences are included between the eukaryotic secretory tag and the GFAP, and between the additional tag and the GFAP.

[0139] In some embodiments of the invention, a linker sequence is included between the eukaryotic secretory tag and the GFAP, and between the additional tag and the GFAP, wherein the additional tag is an affinity tag.

[0140] In some embodiments of the invention, a linker sequence is included between the eukaryotic secretory tag and the GFAP, and between the additional tag and the GFAP, wherein the additional tag is a His tag.

[0141] In some embodiments of the invention, at least one adapter sequence contains a protease cleavage site. The adapter used according to the invention may contain a protease cleavage site, such as a furin protease cleavage site. An advantage is that any tag fused via an adapter having a protease cleavage site can be removed, for example, during or after purification, and therefore may not interfere with the subsequent use 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 protease cleavage site, an 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 an HRV 3C protease cleavage site.

[0143] In some embodiments of the invention, at least one adapter sequence comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. The adapter sequence of SEQ ID NO: 11 comprises a furin cleavage site. In one embodiment, the amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 11 comprises a functional furin cleavage site, i.e., a furin cleavage site capable of being cleaved by furin.

[0144] In some embodiments of the invention, at least one adapter sequence comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

[0145] In some embodiments of the invention, at least one adapter sequence comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. The adapter sequence of SEQ ID NO: 11 contains a furin cleavage site. In one embodiment, the amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 11 contains a functional furin cleavage site, i.e., a furin cleavage site capable of being cleaved by furin.

[0146] In some embodiments of the invention, at least one adapter 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 invention, the protein comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and variants thereof, the variant having at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23.

[0148] In some embodiments of the invention, the protein comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23.

[0149] In some embodiments of the invention, the protein comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and variants thereof, which have at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23.

[0150] In some embodiments of the invention, the protein consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23.

[0151] In a second aspect, the present invention relates to a protein comprising: (i) a GFAP, wherein the GFAP comprises at least 250 consecutive amino acids of an amino acid sequence from SEQ ID NO: 1 or a variant of the amino acid sequence having at least 90% sequence identity with it, and (ii) At least a portion of eukaryotic secretory proteins.

[0152] The proteins in the second aspect, and a portion of the eukaryotic secretory proteins therein, typically lack any signal peptides from the pre-eukaryotic proteins because the signal peptides are cleaved during secretion. A specific example of a portion of a eukaryotic secretory protein is albumin domain 1.

[0153] All embodiments mentioned above in connection with the first aspect of the present invention are applicable to the second aspect of the present invention, and vice versa.

[0154] In some embodiments of the present invention, the protein of the present invention is a recombinant protein.

[0155] In some embodiments of the present invention, the protein is an isolated protein.

[0156] In some embodiments of the present invention, the GFAP comprises at least 260, at least 270, at least 280, at least 290, or at least 300 consecutive amino acids of the amino acid sequence of SEQ ID NO: 1 or a variant of the same amino acid sequence having at least 90% sequence identity. In some embodiments of the present invention, the GFAP comprises at least 300 consecutive amino acids of the amino acid sequence of SEQ ID NO: 1 or a variant of the same amino acid sequence having at least 90% sequence identity. In some embodiments of the present invention, the GFAP comprises at least 309 consecutive amino acids of the amino acid sequence of SEQ ID NO: 1 or a variant of the same amino acid sequence having at least 90% sequence identity.

[0157] In some embodiments of the present invention, the GFAP comprises at least 260, at least 270, at least 280, at least 290, or at least 300 consecutive amino acids of the amino acid sequence of SEQ ID NO: 1 or a variant of the same amino acid sequence having at least 95% sequence identity. In some embodiments of the present invention, the GFAP comprises at least 300 consecutive amino acids of the amino acid sequence of SEQ ID NO: 1 or a variant of the same amino acid sequence having at least 95% sequence identity. In some embodiments of the present invention, the GFAP comprises at least 309 consecutive amino acids of the amino acid sequence of SEQ ID NO: 1 or a variant of the same amino acid sequence having at least 95% sequence identity.

[0158] In some embodiments of the present invention, the GFAP comprises at least 260, at least 270, at least 280, at least 290, or at least 300 consecutive amino acids from the amino acid sequence of SEQ ID NO: 1. In some embodiments of the present invention, the GFAP comprises at least 300 consecutive amino acids from the amino acid sequence of SEQ ID NO: 1. In some embodiments of the present invention, the GFAP comprises at least 309 consecutive amino acids from the amino acid sequence of SEQ ID NO: 1.

[0159] In some embodiments of the invention, the protein is missing amino acids 1 to 68 corresponding to SEQ ID NO: 1 and / or amino acids 378 to 432 corresponding to SEQ ID NO: 1. Truncation of GFAP can lead to further increases in stability, solubility, and / or yield.

[0160] In some embodiments of the present invention, the protein is missing amino acids 1 to 68 corresponding to SEQ ID NO: 1 and amino acids 378 to 432 corresponding to SEQ ID NO: 1.

[0161] In some embodiments of the present invention, the protein contains neither the amino acid sequence of SEQ ID NO: 5 nor a fragment of at least 10 consecutive amino acids.

[0162] In some embodiments of the present invention, the protein contains neither the amino acid sequence of SEQ ID NO: 6 nor a fragment of at least 10 consecutive amino acids.

[0163] In some embodiments of the present invention, the protein contains neither the amino acid sequence of SEQ ID NO: 5 nor a fragment of at least 5 consecutive amino acids.

[0164] In some embodiments of the present invention, the protein contains neither the amino acid sequence of SEQ ID NO: 6 nor a fragment of at least 5 consecutive amino acids.

[0165] In some embodiments of the present invention, the protein contains neither the amino acid sequence of SEQ ID NO: 5 nor a fragment of at least three consecutive amino acids.

[0166] In some embodiments of the present invention, the protein contains neither the amino acid sequence of SEQ ID NO: 6 nor a fragment of at least three consecutive amino acids.

[0167] In some embodiments of the present invention, proteins

[0168] (i) It does not contain the amino acid sequence of SEQ ID NO: 5, nor does it contain a fragment of at least 10 consecutive amino acids therein, and / or

[0169] (ii) It does not contain the amino acid sequence of SEQ ID NO: 6, nor does it contain a fragment of at least 10 consecutive amino acids.

[0170] In some embodiments of the present invention, the protein does not contain 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 the following sequence: (i) SEQ ID NO: 5 and / or (ii) SEQ ID NO: 6.

[0171] In some embodiments of the present invention, GFAP comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof, the variant having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1.

[0172] In some embodiments of the present invention, GFAP comprises the amino acid sequence of SEQ ID NO: 1.

[0173] In some embodiments of the present invention, the amino acid sequence of GFAP consists of SEQ ID NO: 1 or a variant thereof, which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1.

[0174] In some embodiments of the present invention, the amino acid sequence of GFAP consists of SEQ ID NO: 1.

[0175] In some embodiments of the present invention, the amino acid sequence of GFAP consists of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or a variant thereof, the variant having at least 85%, at least 90% or at least 95% sequence identity with SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0176] In some embodiments of the present invention, the amino acid sequence of GFAP consists of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

[0177] In some embodiments of the present invention, the amino acid sequence of GFAP consists of SEQ ID NO: 2 or a variant thereof, which has at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 2.

[0178] In some embodiments of the present invention, the amino acid sequence of GFAP consists of SEQ ID NO: 2.

[0179] In some embodiments of the invention, the protein comprises a portion of a eukaryotic secretory protein.

[0180] In some embodiments of the present invention, the eukaryotic secretion tag comprises a signal peptide derived from a preprotein of a eukaryotic secretory protein.

[0181] In some embodiments of the invention, the eukaryotic secretion tag is a preprotein of a eukaryotic secretory protein or a fragment thereof, wherein the fragment comprises a signal peptide and at least one additional amino acid of the preprotein.

[0182] In some embodiments of the present invention, a portion of the eukaryotic secretory protein is a domain of the eukaryotic secretory protein, i.e., a protein domain. In some embodiments of the present invention, the domain is a functional domain of the eukaryotic secretory protein, which performs the function that the domain normally performs in the context of a full-length eukaryotic secretory protein.

[0183] In some embodiments of the present invention, the protein comprises a full-length eukaryotic secretory protein.

[0184] In some embodiments of the present invention, the eukaryotic secretory protein is selected from the group consisting of albumin domain 1 (AD1 domain), albumin and serum transferrin.

[0185] In some embodiments of the present invention, the eukaryotic secretory protein, or a portion thereof, is derived from mammalian proteins. In some embodiments of the present invention, the eukaryotic secretory protein, or a portion thereof, is derived from mice, rats, goats, African clawed toad Or human eukaryotic secretory proteins. Typically, the amino acid sequence of eukaryotic secretory proteins or a portion thereof is derived from human eukaryotic secretory proteins.

[0186] In some embodiments of the 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 a sequence having at least 85%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0187] In some embodiments of the invention, the eukaryotic secretory protein comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

[0188] In some embodiments of the present invention, the eukaryotic secretory protein is composed of an amino acid sequence selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and a sequence having at least 85%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0189] 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.

[0190] In some embodiments of the invention, the protein contains a eukaryotic secretory protein or a portion thereof at its N-terminus.

[0191] In some embodiments of the present invention, the protein further comprises at least one tag.

[0192] In some embodiments of the present invention, at least one label is a fluorescent label, an affinity label, and / or a solubilizing label.

[0193] In some embodiments of the present invention, at least one tag is a His tag, a FLAG tag, a HA tag, an Avi tag, a strep tag, a MBP tag, an Fc tag, a GFP tag, or a GST tag.

[0194] In some embodiments of the present invention, at least one tag is located at the N-terminus or C-terminus of the GFAP.

[0195] In some embodiments of the present invention, at least one tag is located at the C-terminus of the protein.

[0196] In some embodiments of the invention, the protein of the second aspect comprises a eukaryotic secretory protein or a portion thereof at its N-terminus and an affinity tag at its C-terminus.

[0197] In some embodiments of the invention, the protein of the second aspect comprises a eukaryotic secretory protein or a portion thereof at its N-terminus and a His tag at its C-terminus.

[0198] In some embodiments of the invention, an adapter sequence is included between GFAP and a portion thereof, and optionally between at least one tag and GFAP.

[0199] In some embodiments of the invention, adapter sequences are included between the eukaryotic secretory protein or a portion thereof and GFAP, and between the tag and GFAP, wherein the tag is an affinity tag, typically a His tag.

[0200] In some embodiments of the present invention, at least one adapter sequence contains a protease cleavage site.

[0201] In some embodiments of the present invention, the protease cleavage site is a TEV protease cleavage site, a furin protease cleavage site, an 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 an HRV 3C protease cleavage site.

[0202] In some embodiments of the present invention, the polydispersity index of the protein containing GFAP in solution is at most 1.1. In some embodiments of the present invention, the polydispersity index of the protein containing GFAP in solution is at most 1.05. In some embodiments of the present invention, the polydispersity index of the protein containing GFAP in solution is at most 1.01.

[0203] The polydispersity index can be measured using methods known to those skilled in the art. In particular, the polydispersity index can be measured using MALS (multi-angle static light scattering). In some embodiments of the invention, the light scattering instrument can be coupled to size exclusion chromatography.

[0204] In some embodiments of the invention, the solution used to measure protein polydispersity is an aqueous solution. Typically, a solution containing buffers, salts, and / or other components such as nonionic surfactants, chelating agents, or sodium azide is used.

[0205] In some embodiments of the invention, the protein comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and variants thereof, said variants having at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29.

[0206] In some embodiments of the invention, the protein comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29.

[0207] In some embodiments of the invention, the protein comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and variants thereof, said variants having at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29.

[0208] In some embodiments of the invention, the protein consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29.

[0209] In a third aspect, the present invention relates to nucleic acids encoding proteins of the second aspect.

[0210] All embodiments mentioned in the first and / or second aspects of this invention, with necessary modifications, are applicable to the third aspect of this invention.

[0211] In a fourth aspect, the present invention relates to expression constructs comprising nucleic acids of the first or third aspect.

[0212] All embodiments mentioned in the first, second and / or third aspects of this invention, with necessary modifications, are applicable to the fourth aspect of this invention.

[0213] In some embodiments of the invention, the expression construct 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 invention, the expression construct may further comprise an enhancer to enhance expression. The promoter, terminator, enhancer, 5' UTR, and / or 3' UTR may be operatively linked to the nucleic acid of the invention contained in the expression construct.

[0214] In some embodiments of the invention, the promoter is an inducible promoter. Examples of such inducible promoters include the lac promoter or the tet (tetracycline) promoter with a tetracycline responsive element.

[0215] 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.

[0216] In some embodiments of the present invention, the expression construct includes a human cytomegalovirus immediate early enhancer / promoter region.

[0217] In some embodiments of the present invention, the expression construct includes a bovine growth hormone polyadenylation signal.

[0218] In a fifth aspect, the present invention relates to a carrier comprising the expression construct of the fourth aspect.

[0219] All embodiments mentioned in the first, second, third and / or fourth aspects of this invention, with necessary modifications, are applicable to the fifth aspect of this invention.

[0220] In some embodiments of the invention, the vector of the invention further comprises at least one selection marker and / or at least one origin of replication. The at least one selection marker can select cells containing the vector of the invention. In particular, it can confuse the cell with an advantage over cells that do not contain a vector having at least one selection marker. Examples of selection markers include antibiotic resistance selection markers, which confuse resistance to originally toxic substances, such as β-lactamases or hygromycin B phosphotransferases, which confuse eukaryotic and prokaryotic antibiotic resistance, or auxotrophic selection markers, which confuse the ability to grow in media lacking essential growth substances. Selection markers, also called selectable markers, may be specific to prokaryotic or eukaryotic cells, or may be selection markers for both prokaryotic and eukaryotic cells. The at least one origin of replication can be a eukaryotic or prokaryotic origin of replication. Examples of origins of replication include pSC101, ColE1, pBR322, and pACYC177.

[0221] In some embodiments of the present invention, the vector is a viral vector, an artificial chromosome, or a plasmid.

[0222] In a sixth aspect, the present invention relates to eukaryotic cells comprising the nucleic acid of the first or third aspect, the expression construct of the fourth aspect, or the vector of the fifth aspect.

[0223] All embodiments mentioned in the first, second, third, fourth and / or fifth aspects of this invention, with necessary modifications, are applicable to the sixth aspect of this invention.

[0224] In some embodiments, the nucleic acids, expression constructs, or vectors of the present invention can be integrated into the genome of eukaryotic cells or can exist as extrachromosomal DNA. Eukaryotic cells can transiently contain the nucleic acids, expression constructs, or vectors of the present invention.

[0225] In another embodiment of the invention, nucleic acids, expression constructs, or vectors are integrated into the genome of a eukaryotic cell, i.e., into the chromosome. Integration can be specific or random. Exemplary integration patterns include transposons, such as Sleeping Beauty transposons, or via retroviruses.

[0226] In some embodiments of the present invention, the eukaryotic cells are insect or mammalian cells.

[0227] In some embodiments of the present invention, the eukaryotic cells are mice, rats, goats, etc. African clawed toad Or human cells. In some embodiments of the present invention, eukaryotic cells are human cells.

[0228] In some embodiments of the present invention, the eukaryotic cells are selected from the group consisting of: HeLa cells, CHO cells, Sf21 cells, and Pichia pastoris (…). Pichia pastoris The eukaryotic cells are CHO cells and HEK293 cells. In some embodiments of the invention, the eukaryotic cells are CHO cells or HEK293 cells. In some embodiments of the invention, the eukaryotic cells are HEK293 cells.

[0229] In a seventh aspect, the present invention relates to a method for producing a protein containing GFAP, particularly a GFAP-containing protein of the second aspect, comprising: (a) Provides eukaryotic cells for the sixth aspect, (b) Culture eukaryotic cells in cell culture medium under the following conditions: allowing cells to express and secrete a protein containing GFAP encoded by a nucleic acid of the first or third aspect into the cell culture medium; and thereby producing a protein containing GFAP, and (c) Obtain the protein containing GFAP produced in step (b).

[0230] All embodiments mentioned in the first, second, third, fourth, fifth and / or sixth aspects of this invention, with necessary modifications, are applicable to the seventh aspect of this invention.

[0231] Eukaryotic cells can be provided in step (a) by adding the cells to a cell culture medium. This can be done in ways known to those skilled in the art. For example, the cells can be added in suspension to a similar or different cell culture medium, wherein the cells have been grown prior to addition, or a reserve, such as a glycerol reserve, can be added to the cell culture medium.

[0232] Conditions that allow cells to express and secrete the encoded protein may include: the presence of an inducer in cases where expression is controlled by an inducible promoter; or may include culturing cells under conditions that allow cells to express genes controlled by constitutive promoters. In the case of using an inducer to control expression in step (b), cells may be cultured to the desired cell density before adding the inducer.

[0233] The GFAP-containing protein produced in step (b) can be obtained by any method known to those skilled in the art. In particular, it can be obtained by collecting cell culture medium and optionally including centrifugation and / or filtration steps.

[0234] In some embodiments of the invention, in step (a), eukaryotic cells are provided by adding cells to a cell culture medium.

[0235] In some embodiments of the present invention, the method further includes

[0236] (d) The protein containing GFAP obtained in step (c) is purified.

[0237] The purification in step (d) may include any method known to those skilled in the art suitable for purifying proteins. In some embodiments of the invention, the protein is purified from the cell culture medium by affinity chromatography, particularly immobilized metal affinity chromatography. In some embodiments of the invention, the cell culture medium is centrifuged and / or filtered prior to purification in step (d). The method of the invention may include further purification steps, such as size exclusion chromatography.

[0238] In some embodiments of the invention, eukaryotic secretory proteins or portions thereof, or optionally the additional tag, are cleaved from the GFAP-containing protein during purification.

[0239] In some embodiments of the invention, the eukaryotic secretory protein, or a portion thereof, is cleaved from the protein containing GFAP during purification. In this embodiment, an adapter sequence containing a protease cleavage site is typically included between the eukaryotic secretory tag and the GFAP.

[0240] In some embodiments of the invention, an additional tag is cleaved from the GFAP-containing protein during purification. In this embodiment, an adapter sequence containing a protease cleavage site is typically included between the additional tag and the GFAP.

[0241] In some embodiments of the invention, during secretion into the cell culture medium in step (b), the signal peptide of the eukaryotic secretion tag is cleaved from the protein containing GFAP.

[0242] In some embodiments of the invention, the GFAP-containing protein obtained in step (c) or (d) of the method of the invention is lyophilized in a further step (e). This GFAP-containing lyophilized protein can then be rehydrated.

[0243] In an eighth aspect, the present invention relates to proteins comprising GFAP, which can be obtained, in particular, by the method described in the seventh aspect.

[0244] All embodiments mentioned in the first, second, third, fourth, fifth, sixth and / or seventh aspects of this invention, with necessary modifications, are applicable to the eighth aspect of this invention.

[0245] In a ninth aspect, the present invention relates to a composition comprising the protein of the second or eighth aspect.

[0246] All embodiments mentioned in the first, second, third, fourth, fifth, sixth, seventh and / or eighth aspects of this invention, with necessary modifications, are applicable to the ninth aspect of this invention.

[0247] In some embodiments of the present invention, the composition is a lyophilized product or a solution.

[0248] In some embodiments of the present invention, the composition is an aqueous solution.

[0249] In some embodiments of the present invention, the composition is a lyophilized product. The lyophilized composition of the present invention, i.e., the lyophilized product, has the advantages of being able to be stored under milder conditions compared to, for example, solutions, because the proteins are less susceptible to chemical degradation or hydrolysis. Solutions of the composition may need to be stored at lower temperatures, such as 4°C, but not too low, such as below 0°C, or at freezing temperatures, to extend shelf life without damaging the proteins due to freezing. Simultaneously, the lyophilized composition can be stored under the freezing conditions that other components of the kit containing the lyophilized product may require, because the lyophilized product contains little or no water, thus preventing damage caused by water freezing.

[0250] In some embodiments of the invention, the composition is a lyophilized product, and at least 70% of the GFAP-containing protein of the invention in the composition is reconstituted upon resolution in deionized water.

[0251] In some embodiments of the invention, the composition is a lyophilized product, and at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the GFAP-containing protein of the invention in the composition can be reconstituted upon rehydration in deionized water.

[0252] In some implementations, the percentage of reconstructed proteins containing GFAP is determined by the following method: (A) Prepare a solution containing a protein containing GFAP; (B) A portion of the solution obtained in step (A) of the freeze-drying process; (C) Dissolve the lyophilized product obtained in step (B) in deionized water; (D) Measure the concentration of GFAP-containing protein in the solutions obtained in steps (A) and (C); and (E) Calculate the percentage of reconstructed protein containing GFAP by comparing the concentration of protein containing GFAP in the solution obtained in step (A) with the concentration of protein containing GFAP in the solution obtained in step (C).

[0253] In some implementations, the percentage of reconstructed proteins containing GFAP is determined by the following method: (A) Prepare a solution containing a protein containing GFAP; (B) A portion of the solution obtained in step (A) of the freeze-drying process; (C) Dissolve the lyophilized product obtained in step (B) in deionized water; (D) Measure the amount of GFAP protein contained in the solution obtained in steps (A) and (C); (E) Calculate the amount of GFAP protein in the lyophilized product obtained in step (B) using the amount of GFAP protein in the solution obtained in step (A) and the volume of the solution used in step (B); and (F) Calculate the percentage of reconstructed protein containing GFAP by comparing the amount of GFAP-containing protein in the lyophilized product obtained in step (B) with the amount of GFAP-containing protein in the solution obtained in step (C).

[0254] The concentration of the protein containing GFAP can be measured in step (D) by methods known to those skilled in the art. In some embodiments of the invention, in step (D), the concentration is measured by mass spectrometry or immunoassay, particularly sandwich immunoassay.

[0255] In some embodiments of the invention, the deionized water used to dissolve the lyophilized material in step (c) has a conductivity of less than 1 µS / cm at 25 °C. In some embodiments, the deionized water is Class 2 water according to ISO 3696:1987.

[0256] In a tenth aspect, the present invention relates to a kit comprising: a protein of the second or eighth aspect, or at least a composition of the ninth aspect.

[0257] All embodiments mentioned in the first, second, third, fourth, fifth, sixth, seventh, eighth and / or ninth aspects of this invention, with necessary modifications, are applicable to the tenth aspect of this invention.

[0258] In some embodiments of the present invention, the kit of the present invention contains at least two compositions of the present invention with different concentrations of the proteins of the present invention in separate containers.

[0259] In some embodiments of the invention, the kit is used to calibrate GFAP or its variants in a sample. in vitro Quantitative measurement system.

[0260] The kit of this invention can be used for calibration assays, such as immunoassays. Calibration can be a novel calibration using multiple elevated concentrations of GFAP calibrators, or a single-point or two-point calibration by measuring one or two concentrations of the calibrator. In single-point or two-point calibration, the assay is calibrated using the measured calibrator concentration by correlating the measured concentration with a previously prepared master calibration curve.

[0261] In some embodiments of the invention, the kit contains the composition of the invention in a container. Suitable containers for solid or liquid compositions are known to those skilled in the art and may be glass, optionally with a plastic cap, or plastic.

[0262] In some embodiments of the present invention, the kit comprises multiple containers, each containing the composition of the present invention having the same concentration of the protein of the present invention.

[0263] In some embodiments of the invention, the kit comprises a first container, a second container, and optionally another container, each of which contains a composition of the invention, and the concentration of the protein of the invention contained in each composition in the first, second, and optionally other containers differs from the concentration of GFAP-containing protein in the other containers. For example, the concentration of GFAP-containing protein in the composition in the first container is different from the concentration of GFAP-containing protein in the composition in the second container.

[0264] In this embodiment, the kit may include more than one first container, second container, and / or additional container, wherein each first container, second container, and additional container contains the same concentration of the protein of the present invention.

[0265] In the eleventh aspect, the present invention relates to proteins of the second or eighth aspect, compositions of the ninth aspect, or kits of the tenth aspect for calibrating GFAP or its variants in samples. in vitro Applications of quantitative measurement systems.

[0266] All embodiments mentioned in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and / or tenth aspects of the present invention, with necessary modifications, are applicable to the eleventh aspect of the present invention.

[0267] In some embodiments of the present invention, the sample is a biological fluid sample.

[0268] In some embodiments of the invention, the biofluid sample is whole blood (particularly capillary or venous blood), serum, plasma, saliva, or cerebrospinal fluid. In some embodiments of the invention, the biofluid sample is whole blood. In some embodiments of the invention, the biofluid sample is serum. In some embodiments of the invention, the biofluid sample is plasma.

[0269] In a twelfth aspect, the present invention relates to a method for calibrating GFAP or its variants in a sample. in vitro A method for quantitative measurement system, comprising the step of: using the measurement system to perform at least one measurement on a composition containing at least one GFAP protein according to the ninth aspect and having a known concentration.

[0270] All embodiments mentioned in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth and / or eleventh aspects of the present invention, with necessary modifications, are applicable to the twelfth aspect of the present invention.

[0271] In a further embodiment, the present invention relates to the following: 1. A nucleic acid encoding a protein, said protein comprising... (i) Glial fibrous acidic protein (GFAP), wherein the GFAP comprises at least 250 consecutive amino acids of the amino acid sequence of SEQ ID NO: 1 or a variant of the amino acid sequence having at least 90% sequence identity with it, and (ii) Eukaryotic secretion tags.

[0272] 2. The nucleic acid as described in item 1, wherein the nucleic acid is DNA.

[0273] 3. The nucleic acid according to item 1 or 2, wherein the amino acid sequence of GFAP is derived from human GFAP.

[0274] 4. The nucleic acid according to any one of items 1 to 3, wherein the GFAP comprises at least 260, at least 270, at least 280, at least 290, at least 300 or at least 309 consecutive amino acid bodies of an amino acid sequence from SEQ ID NO: 1 or a variant of the amino acid sequence having at least 90% sequence identity with it.

[0275] 5. The nucleic acid according to any one of items 1 to 3, wherein the GFAP comprises at least 300 consecutive amino acids of an amino acid sequence from SEQ ID NO: 1 or a variant of the amino acid sequence having at least 95% sequence identity with it.

[0276] 6. The nucleic acid according to any one of items 1 to 3, wherein the GFAP comprises at least 300 consecutive amino acids from the amino acid sequence of SEQ ID NO: 1.

[0277] 7. The nucleic acid according to any one of items 1 to 6, wherein the protein is deficient in amino acids 1 to 68 corresponding to amino acids SEQ ID NO: 1 and / or amino acids 378 to 432 corresponding to amino acids SEQ ID NO: 1.

[0278] 8. The nucleic acid according to any one of items 1 to 6, wherein the protein is deficient in amino acids 1 to 68 corresponding to amino acids SEQ ID NO: 1 and amino acids 378 to 432 corresponding to amino acids SEQ ID NO: 1.

[0279] 9. Nucleic acid according to item 7 or 8, wherein the protein

[0280] (i) It does not contain the amino acid sequence of SEQ ID NO: 5, nor does it contain a fragment of at least 10 consecutive amino acids therein, and / or

[0281] (ii) It does not contain the amino acid sequence of SEQ ID NO: 6, nor does it contain a fragment of at least 10 consecutive amino acids.

[0282] 10. The nucleic acid according to any one of items 1 to 6, wherein the GFAP comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof, the variant having at least 75% sequence identity with SEQ ID NO: 1.

[0283] 11. The nucleic acid according to any one of items 1 to 6, wherein the GFAP comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof, the variant having at least 80% sequence identity with SEQ ID NO: 1.

[0284] 12. The nucleic acid according to any one of items 1 to 6, wherein the GFAP comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof, the variant having at least 85% sequence identity with SEQ ID NO: 1.

[0285] 13. The nucleic acid according to any one of items 1 to 6, wherein the GFAP comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof, the variant having at least 90% sequence identity with SEQ ID NO: 1.

[0286] 14. The nucleic acid according to any one of items 1 to 6, wherein the GFAP comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof, the variant having at least 95% sequence identity with SEQ ID NO: 1.

[0287] 15. The nucleic acid according to any one of items 1 to 6, wherein the GFAP comprises the amino acid sequence of SEQ ID NO: 1.

[0288] 16. The nucleic acid according to any one of items 1 to 6, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 1 or a variant thereof, the variant having at least 70% sequence identity with SEQ ID NO: 1.

[0289] 17. The nucleic acid according to any one of items 1 to 6, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 1 or a variant thereof, the variant having at least 75% sequence identity with SEQ ID NO: 1.

[0290] 18. The nucleic acid according to any one of items 1 to 6, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 1 or a variant thereof, the variant having at least 80% sequence identity with SEQ ID NO: 1.

[0291] 19. The nucleic acid according to any one of items 1 to 6, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 1 or a variant thereof, the variant having at least 85% sequence identity with SEQ ID NO: 1.

[0292] 20. The nucleic acid according to any one of items 1 to 6, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 1 or a variant thereof, the variant having at least 90% sequence identity with SEQ ID NO: 1.

[0293] 21. The nucleic acid according to any one of items 1 to 6, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 1 or a variant thereof, the variant having at least 95% sequence identity with SEQ ID NO: 1.

[0294] 22. The nucleic acid according to any one of items 1 to 6, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 1.

[0295] 23. The nucleic acid according to any one of items 1 to 9, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or a variant thereof, the variant having at least 85% sequence identity with SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0296] 24. The nucleic acid according to any one of items 1 to 9, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or a variant thereof, the variant having at least 90% sequence identity with SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0297] 25. The nucleic acid according to any one of items 1 to 9, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or a variant thereof, the variant having at least 95% sequence identity with SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0298] 26. The nucleic acid according to any one of items 1 to 9, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

[0299] 27. The nucleic acid according to any one of items 1 to 9, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 2 or a variant thereof, the variant having at least 85% sequence identity with SEQ ID NO: 2.

[0300] 28. The nucleic acid according to any one of items 1 to 9, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 2 or a variant thereof, the variant having at least 90% sequence identity with SEQ ID NO: 2.

[0301] 29. The nucleic acid according to any one of items 1 to 9, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 2 or a variant thereof, the variant having at least 95% sequence identity with SEQ ID NO: 2.

[0302] 30. The nucleic acid according to any one of items 1 to 9, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 2.

[0303] 31. The nucleic acid according to any one of items 1 to 30, wherein the eukaryotic secretion tag comprises a signal peptide from a preprotein of a eukaryotic secretory protein.

[0304] 32. The nucleic acid according to item 31, wherein the eukaryotic secretion tag consists of a signal peptide derived from a preprotein of a eukaryotic secretory protein.

[0305] 33. The nucleic acid according to any one of items 1 to 31, wherein the eukaryotic secretion tag is a preprotein or fragment of a eukaryotic secretory protein, wherein the fragment comprises a signal peptide and at least one additional amino acid.

[0306] 34. The nucleic acid according to any one of items 1 to 33, wherein the eukaryotic secretory tag is selected from the group consisting of: insulin signal peptide, albumin domain 1 (AD1 domain), albumin and serum transferrin.

[0307] 35. The nucleic acid according to any one of items 1 to 34, wherein the eukaryotic secretory tag is derived from mammalian proteins, particularly human proteins.

[0308] 36. The nucleic acid according to any one of claims 1 to 35, wherein the eukaryotic secretory tag comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

[0309] 37. The nucleic acid according to any one of claims 1 to 35, wherein the eukaryotic secretory tag comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

[0310] 38. The nucleic acid according to any one of items 1 to 35, wherein the eukaryotic secretion tag comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

[0311] 39. The nucleic acid according to any one of items 1 to 35, wherein the eukaryotic secretion tag comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

[0312] 40. The nucleic acid according to any one of items 1 to 39, wherein the protein contains the eukaryotic secretory tag at its N-terminus.

[0313] 41. The nucleic acid according to any one of items 1 to 40, wherein the protein further comprises at least one additional tag.

[0314] 42. The nucleic acid according to item 41, wherein at least one additional tag is a fluorescent tag, an affinity tag, and / or a solubility-enhancing tag.

[0315] 43. The nucleic acid according to item 41 or 42, wherein at least one additional tag is a His tag, a FLAG tag, a HA tag, an Avi tag, a strep tag, a MBP tag, an Fc tag, a GFP tag, or a GST tag.

[0316] 44. The nucleic acid according to any one of items 41 to 43, wherein at least one additional tag is located at the N-terminus or C-terminus of the GFAP.

[0317] 45. The nucleic acid according to any one of items 41 to 44, wherein at least one additional tag is located at the C-terminus of the protein.

[0318] 46. ​​The nucleic acid according to any one of items 41 to 45, wherein the protein contains a eukaryotic secretory tag at its N-terminus and an affinity tag at its C-terminus.

[0319] 47. The nucleic acid according to any one of items 41 to 45, wherein the protein contains a eukaryotic secretion tag at its N-terminus and a His tag at its C-terminus.

[0320] 48. The nucleic acid according to any one of items 1 to 47, wherein an adapter sequence is included between the eukaryotic secretory tag and the GFAP, and optionally between another tag and the GFAP.

[0321] 49. The nucleic acid according to item 48, wherein an adapter sequence is included between the eukaryotic secretory tag and the GFAP, and between an additional tag and the GFAP, wherein the additional tag is an affinity tag, typically a His tag.

[0322] 50. The nucleic acid according to item 48 or 49, wherein at least one adapter sequence contains a protease cleavage site.

[0323] 51. The nucleic acid according to item 50, wherein the protease cleavage site is a TEV protease cleavage site, a furin protease cleavage site, an 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 an HRV 3C protease cleavage site.

[0324] 52. The nucleic acid according to any one of claims 48 to 51, wherein at least one adapter sequence comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

[0325] 53. The nucleic acid according to any one of claims 48 to 51, wherein at least one adapter sequence comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

[0326] 54. The nucleic acid according to any one of claims 48 to 51, wherein at least one adapter sequence comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

[0327] 55. The nucleic acid according to any one of items 48 to 51, wherein at least one adapter sequence comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

[0328] 56. The nucleic acid according to any one of items 1 to 9, 31 to 35, or 40 to 51, wherein the protein comprises or is composed of an amino acid sequence selected from or consisting of the group consisting of: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and variants thereof, said variant having at least 85% sequence identity with SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23.

[0329] 57. The nucleic acid according to any one of items 1 to 9, 31 to 35, or 40 to 51, wherein the protein comprises or is composed of an amino acid sequence selected from or consisting of the group consisting of: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and variants thereof, said variant having at least 90% sequence identity with SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23.

[0330] 58. The nucleic acid according to any one of items 1 to 9, 31 to 35, or 40 to 51, wherein the protein comprises or is composed of an amino acid sequence selected from or consisting of the group consisting of: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and variants thereof, said variants having at least 95% sequence identity with SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23.

[0331] 59. The nucleic acid according to any one of items 1 to 9, 31 to 35 or 40 to 51, wherein the protein comprises or is composed of an amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23.

[0332] 60. A protein comprising

[0333] (i) a GFAP, wherein the GFAP comprises at least 250 consecutive amino acids of an amino acid sequence from SEQ ID NO: 1 or a variant of the amino acid sequence having at least 90% sequence identity with it, and

[0334] (ii) At least a portion of eukaryotic secretory proteins.

[0335] 61. The protein according to item 60, wherein the protein is a recombinant protein.

[0336] 62. The protein according to item 60 or 61, wherein the amino acid sequence of GFAP is derived from human GFAP.

[0337] 63. The nucleic acid according to any one of items 60 to 62, wherein the GFAP comprises at least 260, at least 270, at least 280, at least 290, at least 300 or at least 309 consecutive amino acid bodies of an amino acid sequence from SEQ ID NO: 1 or a variant of the amino acid sequence having at least 90% sequence identity with it.

[0338] 64. The nucleic acid according to any one of items 60 to 62, wherein the GFAP comprises at least 300 consecutive amino acids of an amino acid sequence from SEQ ID NO: 1 or a variant of the amino acid sequence having at least 95% sequence identity with it.

[0339] 65. The nucleic acid according to any one of items 60 to 62, wherein the GFAP comprises at least 300 consecutive amino acids from the amino acid sequence of SEQ ID NO: 1.

[0340] 66. The protein according to any one of claims 60 to 65, wherein the protein is deficient in amino acids 1 to 68 corresponding to SEQ ID NO: 1 and / or amino acids 378 to 432 corresponding to SEQ ID NO: 1.

[0341] 67. The protein according to any one of claims 60 to 65, wherein the protein is deficient in amino acids 1 to 68 corresponding to SEQ ID NO: 1 and amino acids 378 to 432 corresponding to SEQ ID NO: 1.

[0342] 68. The protein according to item 66 or 67, wherein the protein

[0343] (i) It does not contain the amino acid sequence of SEQ ID NO: 5, nor does it contain a fragment of at least 10 consecutive amino acids therein, and / or

[0344] (ii) It does not contain the amino acid sequence of SEQ ID NO: 6, nor does it contain a fragment of at least 10 consecutive amino acids.

[0345] 69. The protein according to any one of claims 60 to 65, wherein the GFAP comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof, the variant having at least 75% sequence identity with SEQ ID NO: 1.

[0346] 70. The protein according to any one of claims 60 to 65, wherein the GFAP comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof, the variant having at least 80% sequence identity with SEQ ID NO: 1.

[0347] 71. The protein according to any one of claims 60 to 65, wherein the GFAP comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof, the variant having at least 85% sequence identity with SEQ ID NO: 1.

[0348] 72. The protein according to any one of claims 60 to 65, wherein the GFAP comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof, the variant having at least 90% sequence identity with SEQ ID NO: 1.

[0349] 73. The protein according to any one of claims 60 to 65, wherein the GFAP comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof, the variant having at least 95% sequence identity with SEQ ID NO: 1.

[0350] 74. The protein according to any one of claims 60 to 65, wherein GFAP comprises the amino acid sequence of SEQ ID NO: 1.

[0351] 75. The protein according to any one of claims 60 to 65, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 1 or a variant thereof, the variant having at least 70% sequence identity with SEQ ID NO: 1.

[0352] 76. The protein according to any one of items 60 to 65, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 1 or a variant thereof, the variant having at least 75% sequence identity with SEQ ID NO: 1.

[0353] 77. The protein according to any one of claims 60 to 65, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 1 or a variant thereof, the variant having at least 80% sequence identity with SEQ ID NO: 1.

[0354] 78. The protein according to any one of claims 60 to 65, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 1 or a variant thereof, the variant having at least 85% sequence identity with SEQ ID NO: 1.

[0355] 79. The protein according to any one of claims 60 to 65, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 1 or a variant thereof, the variant having at least 90% sequence identity with SEQ ID NO: 1.

[0356] 80. The protein according to any one of claims 60 to 65, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 1 or a variant thereof, the variant having at least 95% sequence identity with SEQ ID NO: 1.

[0357] 81. The protein according to any one of items 60 to 65, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 1.

[0358] 82. The protein according to any one of claims 60 to 68, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or a variant thereof, the variant having at least 85% sequence identity with SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0359] 83. The protein according to any one of claims 60 to 68, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or a variant thereof, the variant having at least 90% sequence identity with SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0360] 84. The protein according to any one of claims 60 to 68, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or a variant thereof, the variant having at least 95% sequence identity with SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0361] 85. The protein according to any one of items 60 to 68, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

[0362] 86. The protein according to any one of claims 60 to 68, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 2 or a variant thereof, the variant having at least 85% sequence identity with SEQ ID NO: 2.

[0363] 87. The protein according to any one of claims 60 to 68, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 2 or a variant thereof, the variant having at least 90% sequence identity with SEQ ID NO: 2.

[0364] 88. The protein according to any one of claims 60 to 68, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 2 or a variant thereof, the variant having at least 95% sequence identity with SEQ ID NO: 2.

[0365] 89. The protein according to any one of items 60 to 68, wherein the amino acid sequence of GFAP consists of SEQ ID NO: 2.

[0366] 90. The protein according to any one of items 60 to 89, wherein the protein comprises a portion of a eukaryotic secretory protein.

[0367] 91. The protein according to any one of items 60 to 90, wherein the protein comprises a full-length eukaryotic secretory protein.

[0368] 92. The protein according to any one of items 60 to 91, wherein the eukaryotic secretory protein is selected from the group consisting of: albumin domain 1 (AD1 domain), albumin, and serum transferrin.

[0369] 93. The protein according to any one of items 60 to 92, wherein the eukaryotic secretory protein or a portion thereof is derived from mammalian eukaryotic secretory proteins, particularly human eukaryotic secretory proteins.

[0370] 94. The protein according to any one of claims 60 to 93, wherein the eukaryotic secretory protein comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and a sequence having at least 85% sequence identity with any one of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0371] 95. The protein according to any one of claims 60 to 93, wherein the eukaryotic secretory protein comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and a sequence having at least 90% sequence identity with any one of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0372] 96. The protein according to any one of claims 60 to 93, wherein the eukaryotic secretory protein comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and a sequence having at least 95% sequence identity with any one of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0373] 97. The protein according to any one of claims 60 to 93, wherein the eukaryotic secretory protein comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

[0374] 98. The protein according to any one of claims 60 to 97, wherein the protein comprises, at its N-terminus, the eukaryotic secretory protein or a portion thereof.

[0375] 99. The protein according to any one of items 60 to 98, wherein the protein further comprises at least one tag.

[0376] 100. The protein according to item 99, wherein at least one tag is a fluorescent tag, an affinity tag, and / or a solubilizing tag.

[0377] 101. The protein according to item 99 or 100, 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.

[0378] 102. The protein according to any one of claims 99 to 101, wherein at least one tag is located at the N-terminus or C-terminus of the GFAP.

[0379] 103. The protein according to any one of claims 99 to 102, wherein at least one tag is located at the C-terminus of the protein.

[0380] 104. The protein according to any one of items 99 to 103, wherein the protein comprises a eukaryotic secretory protein or a portion thereof at its N-terminus and an affinity tag at its C-terminus.

[0381] 105. The protein according to any one of items 99 to 104, wherein the protein comprises a eukaryotic secretory protein or a portion thereof at its N-terminus and a His tag at its C-terminus.

[0382] 106. The protein according to any one of claims 60 to 105, wherein an adapter sequence is included between the GFAP and the eukaryotic secretory protein or a portion thereof, and optionally between at least one tag and the GFAP.

[0383] 107. The protein according to claim 106, wherein an adapter sequence is included between the eukaryotic secretory protein or a portion thereof and the GFAP, and between the tag and the GFAP, wherein the tag is an affinity tag, typically a His tag.

[0384] 108. The protein according to item 106 or 107, wherein at least one adapter sequence contains a protease cleavage site.

[0385] 109. The protein according to claim 108, wherein the protease cleavage site is a TEV protease cleavage site, a furin protease cleavage site, an 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 an HRV 3C protease cleavage site.

[0386] 110. The protein according to any one of claims 60 to 109, wherein the protein containing GFAP, when in solution, has a polydispersity index of at most 1.1, typically at most 1.05, and more typically at most 1.01.

[0387] 111. The protein according to any one of claims 106 to 110, wherein at least one adapter sequence comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

[0388] 112. The protein according to any one of claims 106 to 110, wherein at least one adapter sequence comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

[0389] 113. The protein according to any one of claims 106 to 110, wherein at least one adapter sequence comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

[0390] 114. The protein according to any one of claims 106 to 110, wherein at least one adapter sequence comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

[0391] 115. The protein according to any one of claims 60 to 68, 90 to 93, or 98 to 110, wherein the protein comprises or is composed of an amino acid sequence selected from or consisting of the group consisting of: SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and variants thereof, said variants having at least 85% sequence identity with SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29.

[0392] 116. The protein according to any one of claims 60 to 68, 90 to 93, or 98 to 110, wherein the protein comprises or is composed of an amino acid sequence selected from or consisting of the group consisting of: SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and variants thereof, said variants having at least 90% sequence identity with SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29.

[0393] 117. The protein according to any one of claims 60 to 68, 90 to 93, or 98 to 110, wherein the protein comprises or is composed of an amino acid sequence selected from or consisting of the group consisting of: SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and variants thereof, said variants having at least 95% sequence identity with SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29.

[0394] 118. The protein according to any one of claims 60 to 68, 90 to 93, or 98 to 110, wherein the protein comprises or is composed of an amino acid sequence selected from or consisting of the group consisting of: SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29.

[0395] 119. A nucleic acid that encodes a protein according to any one of items 60 to 118.

[0396] 120. An expression construct comprising any one of claims 1 to 59 or 119.

[0397] 121. The expression construct according to item 120, comprising a promoter, a terminator, a 5' UTR, and / or a 3' UTR.

[0398] 122. The expression construct according to item 121, wherein the promoter is an inductive promoter.

[0399] 123. The expression construct according to item 122, wherein the promoter is a constitutive promoter.

[0400] 124. The expression construct according to item 120 or 122, wherein the expression construct contains a human cytomegalovirus immediate early enhancer / promoter region.

[0401] 125. The expression construct according to any one of claims 120 to 124, wherein the expression construct contains bovine growth hormone polyadenylation signal.

[0402] 126. A vector comprising an expression construct according to any one of claims 120 to 125.

[0403] 127. The carrier according to item 126, comprising at least one selection marker and / or at least one origin of replication.

[0404] 128. The vector according to item 126 or 127, wherein the vector is a viral vector, an artificial chromosome, or a plasmid.

[0405] 129. A eukaryotic cell comprising a nucleic acid according to any one of claims 1 to 59 or 119, an expression construct according to any one of claims 120 to 125, or a vector according to any one of claims 126 to 128.

[0406] 130. The eukaryotic cell according to item 129, wherein the eukaryotic cell is a human cell.

[0407] 131. The eukaryotic cell according to item 129 or 130, wherein the eukaryotic cell is a CHO or HEK293 cell.

[0408] 132. A method for producing a protein comprising GFAP, particularly the protein according to any one of claims 60 to 118, the method comprising: (a) Providing eukaryotic cells according to any one of items 129 to 131, (b) The eukaryotic cells are cultured in a cell culture medium under the following conditions: the cells are allowed to express and secrete a protein containing GFAP encoded by a nucleic acid according to any one of items 1 to 59 or 119 into the cell culture medium; and thereby produce a protein containing GFAP, and (c) Obtain the protein containing GFAP produced in step (b).

[0409] 133. The method according to item 132, further comprising:

[0410] (d) The protein containing GFAP obtained in step (c) is purified.

[0411] 134. The method according to item 133, wherein the protein is purified from the cell culture medium by affinity chromatography, particularly immobilized metal affinity chromatography.

[0412] 135. The method according to any one of items 132 to 134, wherein during secretion into the cell culture medium in step (b), the signal peptide of the eukaryotic secretion tag is cleaved from the protein containing GFAP.

[0413] 136. The method according to any one of items 132 to 135, wherein during purification, eukaryotic secretory proteins or portions thereof, or optionally additional tags, are cleaved from the protein containing GFAP.

[0414] 137. The method according to any one of items 132 to 136, wherein the GFAP obtained in step (c) or (d) of the method of the invention is freeze-dried in a further step (e).

[0415] 138. A protein comprising GFAP, which can be obtained by, in particular by, any one of claims 132 to 137.

[0416] 139. A composition comprising the protein according to any one of claims 60 to 118 or 138.

[0417] 140. The composition according to claim 139, wherein the composition is a lyophilized product or a solution, typically a lyophilized product.

[0418] 141. The composition according to claim 139 or 140, wherein the composition is a lyophilized product, and at least 70% of the GFAP-containing protein according to any one of claims 60 to 118 or 138 in the composition is reconstituted upon resolvation in deionized water.

[0419] 142. The composition according to claim 139 or 140, wherein the composition is a lyophilized product, and at least 90% of the GFAP-containing protein in the composition according to any one of claims 60 to 118 or 138 is reconstituted upon resolvation in deionized water.

[0420] 143. The composition according to claim 139 or 140, wherein the composition is a lyophilized product, and at least 95% of the GFAP-containing protein in the composition according to any one of claims 60 to 118 or 138 is reconstituted upon resolvation in deionized water.

[0421] 144. The composition according to claim 139 or 140, wherein the composition is a lyophilized product, and at least 97% of the GFAP-containing protein in the composition according to any one of claims 60 to 118 or 138 is reconstituted upon resolvation in deionized water.

[0422] 145. The composition according to claim 139 or 140, wherein the composition is a lyophilized product, and at least 98% of the GFAP-containing protein according to any one of claims 60 to 118 or 138 in the composition is reconstituted upon resolvation in deionized water.

[0423] 146. The composition according to claim 139 or 140, wherein the composition is a lyophilized product, and at least 99% of the GFAP-containing protein according to any one of claims 60 to 118 or 138 in the composition is reconstituted upon resolvation in deionized water.

[0424] 147. The composition according to any one of items 141 to 146, wherein the percentage of reconstructed protein containing GFAP is determined by the following method.

[0425] (A) Prepare a solution containing a protein containing GFAP; (B) A portion of the solution obtained in step (A) of freeze-drying; (C) Dissolve the lyophilized product obtained in step (B) in deionized water; (D) Measure the concentration of GFAP-containing proteins in the solutions obtained in steps (A) and (C); (E) Calculate the percentage of reconstructed protein containing GFAP by comparing the concentration of protein containing GFAP in the solution obtained in step (A) with the concentration of protein containing GFAP in the solution obtained in step (C).

[0426] 148. The composition according to item 147, wherein in step (D), the concentration is measured by mass spectrometry or immunoassay, particularly sandwich immunoassay.

[0427] 149. A kit comprising: a protein according to any one of claims 60 to 118 or 138, or at least one composition according to any one of claims 139 to 148.

[0428] 150. The kit according to claim 149, comprising a first container, a second container, and optionally a further container, wherein each of the first container, the second container, and the optional further container contains a composition according to any one of claims 139 to 148, and the concentration of the protein according to any one of claims 60 to 118 or 138 contained in each composition of the first container, the second container, and the optional further container is different from the concentration of the GFAP protein contained in the other containers.

[0429] 151. The kit described in item 149 or 150, for calibrating GFAP or its variants in a sample. body outside Quantitative measurement system.

[0430] 152. The protein according to any one of claims 60 to 118 or 138, the composition according to any one of claims 139 to 148, or the kit according to any one of claims 149 to 151 for calibrating GFAP or its variants in a sample. in vitro Applications of quantitative measurement systems.

[0431] 153. The use as described in item 152, wherein the sample is a biological fluid sample.

[0432] 154. The use according to item 153, wherein the biological fluid sample is whole blood, particularly capillary or venous blood, serum, plasma, saliva or cerebrospinal fluid.

[0433] 155. A method for calibrating GFAP or a variant thereof in a sample. in vitro A method for a quantitative measurement system, comprising the steps of: using the measurement system to perform at least one measurement on a composition containing at least one GFAP-containing protein according to any one of items 139 to 148 and having a known concentration.

[0434]

[0435] Example

[0436] The following examples are provided to illustrate, rather than limit, the inventions claimed herein.

[0437] Example 1

[0438] Recombinant expression of GFAP

[0439] For transient gene expression, the FreeStyle™ 293-F expression system (Thermo Fischer Scientific) was used. The following expression construct was designed, containing a human cytomegalovirus (CMV) early enhancer / promoter region followed by the bovine growth hormone polyadenylation signal: GFAP-His8, GFAP_HUMAN, Uniprot P14136 residues 1-432, with a C-terminal His tag. Full-length human glial fibrillary acidic protein GFAP-His8 is an aggregated cytoplasmic protein, making it difficult to express: Transfect approximately 2 × 10⁶ cells / ml of culture with an expression plasmid in combination with PEIpro™ transfection reagent (Polyplus) at a concentration of 0.5 mg / L, following the manufacturer's instructions. 72 hours post-transfection, the cell pellet was collected by centrifugation and stored at -80°C for cell lysis and subsequent protein purification. Frozen cells were thawed and resuspended in 50 mM HEPES (pH 7.5), 300 mM NaCl, 5 mM MgCl₂, 5% glycerol, and 0.1% polysorbate 20. Immediately before use, a protease inhibitor (10 tablets per 100 ml, completely EDTA-free (Roche)) and DNase I were added to the buffer. A Potter-Elvehjem homogenizer was used for cell lysis. For purification, the clarified lysate was applied to an IMAC chromatogram. His-tagged GFAP was eluted with imidazole and dialyzed into a final storage buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 6.5% sucrose).

[0440] Only a very small amount of GFAP-His8 without eukaryotic secretion tags could be purified, while almost all expressed GFAPs had aggregated.

[0441] Example 2

[0442] Next, the secretory expression of GFAP was examined. GFAP is a strictly cytoplasmic protein and therefore not naturally intended to be secreted. Thus, the outcome of GFAP secretion is unpredictable, as it is impossible to predict in advance whether GFAP will actually be secreted, retained in the ER, or proteolytically metabolized. For transient gene expression, QMCF technology (Icosagen) was used. Multiple expression constructs were designed, using different secretion signal / fusion couplers for full-length GFAP (Residues 2-432 of Uniprot P14136 were used for GFAP; the complete sequence is shown below). All coding sequences were cloned into the pQ1TAR expression vector, and the coding regions were verified by sequencing.

[0443] The following expression builder was constructed: Insulin signal peptide GFAP FLAG tag (1), Albumin domain 1-linker GFAP-FLAG tag with signal peptide (2), Albumin with signal peptide linker GFAP FLAG tag (3), The albumin domain 1_linker 2_GFAP_FLAG tag with signal peptide is present (4), and Serum transferrin with signal peptide_connector_GFAP_FLAG tag (5) CHOEBNALT-85-1E9 CHO cells were transiently transfected with expression plasmids via chemical transfection according to the QMCF technology cell processing manual (Icosagen). Transfected cells were cultured in CHO TF (Xell AG) growth medium for up to 10 days, after which the cells were harvested and the supernatant was clarified by filtration or centrifugation. The supernatant was analyzed using Western blotting with a 4% to 15% gradient SDS-polyacrylamide gel using rabbit anti-FLAG (F7425, SigmaAldrich; 0.4 mg / ml, 1:250) and goat anti-rabbit IgG H&L (HRP) (ab97051, Abcam, 1:8000).

[0444] For all expression constructs (1 = insulin signal peptide_GFAP_FLAG tag, approx. 50 kDa; 2 = albumin domain 1_connector_GFAP_FLAG tag with signal peptide, approx. 75 kDa; 3 = albumin_connector_GFAP_FLAG tag with signal peptide, approx. 120 kDa; 4 = albumin domain 1_connector2_GFAP_FLAG tag with signal peptide, approx. 75 kDa; 5 = serum transferrin_connector_GFAP_FLAG tag with signal peptide, approx. 125 kDa), expression products of the expected size were visible (see Figure 1). All fusion constructs surprisingly produced soluble proteins of the expected size, which were secreted into the supernatant.

[0445] Example 3

[0446] Using Elecsys ® GFAP-Assay for determining the concentration of GFAP in the supernatant

[0447] Antibodies against glial fibrillary acidic protein were produced at Roche Diagnostics GmbH (Penzberg, Germany), and immunoglobulin G was purified according to the supplier's manual using protein A chromatography (MabSelect SuRe, GE Healthcare, Uppsala Sweden). At Elecsys... ®A sandwich immunoassay has been developed on the platform. (Elecsys) ® The electrochemiluminescence technology on the platform is based on streptavidin-coated microbeads; the capture antibody is biotinylated, and the detection antibody is ruthenium-labeled to generate a signal, as previously described (Hermanson GT. Bioconjugate techniques: Academic press; 2013). Following the standard procedures of the Elecsys platform, a 30 μl sample volume was determined within a total reaction volume of 120 μl (30 μl sample, 66 μl reagent, and 24 μl magnetic beads), and incubated at 37 °C for 18 minutes. Experiments were performed on the Elecsys platform. ® Performed on the CobasPro system.

[0448] Insulin signaling peptide GFAP FLAG tag (1): 0.44 mg / mL

[0449] Albumin domain 1 linker with signal peptide GFAP FLAG tag (2): 1.85 mg / mL

[0450] Albumin with signal peptide linker GFAP FLAG tag (3): 1.97 mg / mL

[0451] Albumin domain 1-linker 2-GFAP-FLAG tag with signal peptide (4): 2.07 mg / mL

[0452] Serum transferrin with signal peptide linker GFAP FLAG tag (5): 0.16 mg / mL

[0453] Albumin and albumin domain 1 fusion proteins yielded similar results, and insulin and serum transferrin fusion proteins also worked, but at lower yields in production.

[0454] Example 4

[0455] Stability test

[0456] To simulate the long-term stability of the specified GFAP calibrator material, an equal volume of the GFAP construct was spiked into human plasma matrix (ROCHE Mat. No. 1220452001). The GFAP construct was directly obtained from cell culture supernatant (as described above) without further purification. (Based on Elecsys...) ®The required amount was calculated quantitatively (see Example 3). A total of 3000 µl solutions of plasma sample were prepared to a final concentration of approximately 1 ng / ml of the GFAP construct. Parallel samples were stored for a total of 8 days at 4 °C, 20 °C, and 35 °C. The samples were then processed using the above-described Elecsys solution on days 0, 1, 2, 5, and 8. ® The GFAPAssay validated the signal recovery of soluble GFAP protein. Table 1 shows the results (%) relative to day 0.

[0457] Table 1. Stability test - Recovery rate in plasma diluted samples compared to day 0.

[0458] Under all test conditions, the stability of all fusion proteins was similar with almost no loss, and all fusion proteins were stable throughout the test time, even under harsh conditions at 35 °C.

[0459] Example 5

[0460] Then, fragments of GFAP were tested, particularly the secretory expression of truncated GFAP. The truncated GFAP was expressed as an albumin fusion protein for secretion into the cell culture supernatant.

[0461] For transient gene expression, the FreeStyle™ 293-F expression system (Thermo Fischer Scientific) was used. The following expression constructs were designed, each with the same human cytomegalovirus immediate early enhancer / promoter region and the same bovine growth hormone polyadenylation signal following the coding gene region: GFAP-His8, GFAP_HUMAN, Uniprot P14136 residues 1-432, with a C-terminal His tag. ALB (albumin)-GFAP-His8, ALBU_HUMAN, Uniprot P02768 residues 1-609, is fused with GFAP_HUMAN, Uniprot P14136 residues 1-432, and a C-terminal His tag is added. ALB-GFAP_69-377-His8, ALBU_HUMAN, Uniprot P02768 residues 1-609, is fused with GFAP_HUMAN, Uniprot P14136 residues 69-377, and a C-terminal His tag is added. Full-length human glial fibrillary acidic protein GFAP-His8 is an aggregated cytoplasmic protein: Transfect approximately 2 × 10⁶ cells / ml of culture with an expression plasmid at a concentration of 0.5 mg / L in combination with PEIpro™ transfection reagent (Polyplus) according to the manufacturer's guidelines. 72 hours post-transfection, collect the cell pellet by centrifugation and store it at -80°C for cell lysis and subsequent protein purification. Thaw frozen cells and resuspend them in 50 mM HEPES (pH 7.5), 300 mM NaCl, 5 mM MgCl₂, 5% glycerol, and 0.1% polysorbate 20. Immediately before use, add the protease inhibitor (10 tablets per 100 ml, completely EDTA-free (Roche)) and DNase I to the buffer. Use a Potter-Elvehjem homogenizer for cell lysis. For purification, apply the clarified lysate to an IMAC chromatograph. 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).

[0462] In contrast, fusion with the C-terminus of human albumin resulted in the secretion of ALB-GFAP-His8 and ALB-GFAP_69-377-His8 into the HEK293 culture supernatant, facilitating purification: 7 days post-transfection, cell supernatants were collected by centrifugation and stored at -80 °C, followed by protein purification by IMAC chromatography. The His-tagged ALB-GFAP fusion protein was eluted with imidazole and purified using a Superose™ 200 column via preparative size exclusion chromatography.

[0463] When analyzed by mass spectrometry, the purified ALB-GFAP-His8 showed no signs of glycosylation.

[0464] The purified GFAP construct was characterized by SDS-PAGE under reducing conditions using Coomassie blue staining for protein detection.

[0465] For SDS-PAGE analysis, treat approximately 5 µg of protein with NuPAGE™ SDS Sample Buffer (4x) (Invitrogen) containing 100 mM DTT according to the manufacturer's instructions, and apply it to a NuPAGE™ 4-12% Bis-Tris gel (Invitrogen). Use Roti ®Blue Quick (Roth) was used for staining.

[0466] Both proteins exhibited an apparent molecular weight of approximately 100 kDa and good purity in SDS-PAGE (see Figures 2A and 2B). Therefore, it was found that truncated GFAP can also be expressed and secreted from eukaryotic cells.

Claims

1. A nucleic acid encoding a protein, said protein comprising... (i) Glial fibrous acidic protein (GFAP), wherein the GFAP comprises at least 250 consecutive amino acids of the amino acid sequence from SEQ ID NO: 1 or a variant of the amino acid sequence having at least 90% sequence identity with it, and (ii) Eukaryotic secretion tags.

2. The nucleic acid according to claim 1, wherein the GFAP comprises at least 260, at least 270, at least 280, at least 290, at least 300, or at least 309 consecutive amino acids of the amino acid sequence from SEQ ID NO: 1 or a variant of the amino acid sequence having at least 90% sequence identity with it.

3. The nucleic acid according to claim 1 or 2, wherein the eukaryotic secretion tag is a preprotein of a eukaryotic secretory protein or a fragment thereof, wherein the fragment comprises a signal peptide and at least one additional amino acid.

4. The nucleic acid according to any one of claims 1 to 3, wherein the eukaryotic secretory tag is selected from the group consisting of: insulin signal peptide, albumin domain 1 (AD1 domain), albumin, and serum transferrin.

5. A protein comprising (i) GFAP, wherein the GFAP comprises at least 250 consecutive amino acids of the amino acid sequence from SEQ ID NO: 1 or a variant of the amino acid sequence having at least 90% sequence identity with it, and (ii) At least a portion of eukaryotic secretory proteins.

6. The protein of claim 5, wherein the GFAP comprises at least 260, at least 270, at least 280, at least 290, at least 300, or at least 309 consecutive amino acids of the amino acid sequence of SEQ ID NO: 1 or a variant of the amino acid sequence having at least 90% sequence identity with it.

7. The protein according to claim 5 or 6, wherein the eukaryotic secretory protein is selected from the group consisting of: albumin domain 1 (AD1 domain), albumin, and serum transferrin.

8. An expression construct comprising the nucleic acid according to any one of claims 1 to 4.

9. A vector comprising the expression construct according to claim 8.

10. A eukaryotic cell comprising the nucleic acid according to any one of claims 1 to 4, the expression construct according to claim 8, or the vector according to claim 9.

11. A method for producing a protein containing GFAP, the method comprising: (a) Providing the eukaryotic cell according to claim 10, (b) The eukaryotic cells are cultured in a cell culture medium under the following conditions: the cells are allowed to express and secrete the GFAP-containing protein encoded by the nucleic acid according to any one of claims 1 to 4 into the cell culture medium; And thereby producing the protein containing GFAP, and (c) Obtain the GFAP-containing protein produced in step (b).

12. A protein comprising GFAP, which can be obtained, in particular, by the method according to claim 11.

13. A composition comprising the protein according to any one of claims 5 to 7 or 12.

14. A kit comprising: a protein according to any one of claims 5 to 7 or 12, or at least one composition according to claim 13.

15. The protein according to any one of claims 5 to 7 or 12, the composition according to claim 13, or the kit according to claim 14 for calibrating GFAP or its variants in a sample. in vitro Applications of quantitative measurement systems.

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