WO2011072647A1

WO2011072647A1 - Protein or polypeptide for bonding to tubulin and/or microtubule structures

Info

Publication number: WO2011072647A1
Application number: PCT/DE2010/001447
Authority: WO
Inventors: Ralf Erdmann; Wolfgang Schliebs; Carsten Theiss; Alexander Neuhaus
Original assignee: Ralf Erdmann; Wolfgang Schliebs; Carsten Theiss; Alexander Neuhaus
Priority date: 2009-12-15
Filing date: 2010-12-13
Publication date: 2011-06-23
Also published as: DE102009058169A1; DE102009058169B4

Abstract

The invention relates to a protein or polypeptide, comprising an amino acid sequence comprising at least one lysine-rich sequence area, wherein the protein or polypeptide bonds to tubulin.

Description

Protein or polypeptide for binding to tubulin and / or microtubule structures

The invention relates to a protein or polypeptide comprising an amino acid sequence comprising at least one lysine-rich sequence region, wherein the protein or polypeptide binds to tubulin. Preferably, the invention relates to a biomarker for tubulin, as well as to a microtransporter for delivery of drugs into cells, comprising the protein or polypeptide according to claim 1.

In addition to microfilaments and intermediary filaments, microtubules are an important part of the cytoskeleton structure of eukaryotic cells. The cytoskeleton not only performs static, structural tasks, it is also involved in the development and function of individual cell types. The basic building block of the hollow cylindrical microtubules are protofilaments, which are predominantly formed from α- and β-tubulin heterodimers. In the centrosome of the cell also γ-tubulin is expressed, which is involved in the formation of new microtubules in the centrosome region. Microtubules form the framework for intracellular transport processes and are involved in the formation of the spindle apparatus during cell division. The microtubule assembly and disassembly process is very dynamic and centrally controlled by the MTOC (microtubule organizing center). An intact cytoskeleton, especially an intact microtubule structure, is physiologically important in order to ensure the correct transport and uniform distribution of various important proteins and cell organelles. Defects in the functionality of the microtubule structures or their associated proteins can lead to serious diseases such as Alzheimer's disease. In addition, chemotherapeutic agents such as vinblastine or paclitaxel are specifically used to

CONFIRMATION COPY to interfere with the correct assembly of microtubules in cancer cells. An uncontrolled cell division is thereby inhibited. For a better understanding of the cytoskeletal structures and the possible influence of drugs on microtubules, it is of considerable importance to detect cytoskeletal structures, especially microtubule structures, and their changes in cells.

Specific antibodies are already known in the art which can be used to detect tubulin in vitro or in vivo. For example, WO2007 / 095359 A2 discloses the detection of tubulin in cells by the antibodies DM1-a, YL1-2 and 3A2. Many further tubulin-specific antibodies are known to the person skilled in the art. However, the detection of tubulin and thus also microtubule suspensions with the aid of antibodies generally has a considerable disadvantage. The detection of antibodies usually has to be carried out in a multi-step procedure. For this purpose, a tubulin-specific primary antibody is first bound to tubulin. This is then detected in a second step with a directed against the primary antibody, chemically labeled secondary antibody via fluorochromes or other chemical markers. Although specific antibodies can also be directly chemically labeled - for example, with a fluorescent molecule or an enzyme - but this method is very expensive, expensive and usually unsatisfactory in their sensitivity.

There is therefore a need to provide a faster, but also in terms of cost and accuracy of detection optimized detection means for tubulin, or Mikrotubulistrukturen. The above object is achieved by providing the protein or polypeptide of claim 1 of the invention which comprises an amino acid sequence containing at least one lysine-rich Sequence region, wherein the protein or polypeptide binds to tubulin. A lysine-rich sequence region is to be understood as meaning a sequence region which has at least one lysine residue, but preferably a plurality of lysine residues arranged in the immediate vicinity. According to the invention, it is an artificially produced polypeptide or an isolated protein or a subsection isolated therefrom. Surprisingly, it has been found in laboratory studies that the peroxisomal biogenesis factor PEX14 binds to tubulin with high affinity. The investigations showed that the binding region of the protein to tubulin has at least one sequence segment which contains lysine. This sequence section plays a crucial role in the binding of the peroxisomal biogenesis factor PEX14 to tubulin. The protein or polypeptide according to the invention comprises at least one such lysine-rich sequence segment. Advantageously, the protein or polypeptide according to the invention can be biochemically labeled and thus used directly as a biomarker for the detection of tubulin structures. Likewise, the coupling to a medicinal agent is possible, which can reach its site of action in the cell in conjunction with the protein or polypeptide according to the invention.

According to one embodiment, the protein or polypeptide has an amino acid sequence which has at least the sequence Phe-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaaa. Phe-Xaa-Xaa-Xaa-Lys. The two spaced apart phenylalanine residues support the binding affinity of the protein or polypeptide of the invention to tubulin.

According to a further embodiment of the invention, the protein or polypeptide has an amino acid sequence which has at least the sequence Lys-Phe-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa -Xaa-Xaa-Phe-Xaa-Xaa-Xaa-Lys (SEQ ID NO: 1). According to the invention, the binding affinity of the protein or polypeptide is still enhanced by two lysine-containing sequence regions spaced apart from each other elevated. The positively charged lysine residues preferentially bind to strongly negatively charged regions of the tubulin, such as glutamate residues. The invention also binds to a protein or polypeptide

Tubulin, provided it has an amino acid sequence with the sequence Lys-Phe-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Phe-Xaa-Lys Lys-Lys (SEQ ID NO: 2). The invention particularly relates to a protein or polypeptide according to the aforementioned embodiments comprising an amino acid sequence according to SEQ ID NO: 3 or a partial sequence thereof. SEQ ID NO: 3 relates to amino acids 20 to 78 of the N-terminal region of the human peroxisomal biogenesis factor PEX14. According to the invention, a portion of this portion is also sufficient for binding, especially if it has two lysine-rich sequence regions, i. Sequence areas in which at least one lysine residue is present. Likewise, an amino acid sequence according to the invention is suitable provided that it comprises an amino acid sequence of SEQ ID NO: 3 modified by substitution, insertion or addition of amino acids. Preferably, the sequence regions surrounding the lysine residues are rich in the amino acids alanine, threonine and arginine.

The invention also relates to a protein or polypeptide comprising an amino acid sequence according to SEQ ID NO: 4. This sequence relates to amino acids 1-78 of the N-terminal region of the human peroxisomal biogenesis factor PEX14.

Preferably, the protein or polypeptide forms a three-dimensional structure comprising at least three α-helices. Binding to tubulin is optimal when the three α-helices are roughly U-shaped are. They can be stabilized by a fourth diagonal helix. An example of a preferred three-dimensional structure of the protein or polypeptide according to the invention is shown in FIG. According to a further embodiment, the protein or

Polypeptide is present in its binding to tubulin as a dimer. Preferably, the protein or polypeptide is coupled to glutathione-S-transferase (GST). GST promotes the formation of dimers promotes. According to the invention, the dimerization already takes place before the binding to the target protein tubulin. For example, the protein or polypeptide of the present invention is a fusion protein comprising GST and the tubulin-binding amino acid sequence.

The protein or polypeptide has a high dissociation constant according to a preferred embodiment of the invention. The protein or polypeptide according to the invention thus binds to tubulin with high affinity.

In one embodiment of the invention, the protein or polypeptide is coupled to at least one heterologous molecule. The protein or polypeptide can be linked to the heterologous molecule via the C- or the N-terminus of the amino acid chain, as well as via side chains of individual amino acids. A linkage with several similar or mutually different heterologous molecules is possible. For example, the protein or polypeptide of the invention may be present as a fusion protein with the heterologous molecule.

According to the invention, the heterologous molecule can be a marker molecule. This may have fluorescent properties, such as FITC (fluorescein isothiocyanate). A fluorescent marker molecule is preferably a polypeptide, for example GFP (green fluorescent protein), YFP (yellow fluorescent protein), CFP (cyan fluorescent protein), BFP (blue fluorescent protein) or drFP583 (red fluorescent protein). For example, the protein or polypeptide of the invention may be present as a fusion protein, being linked to GFP or YFP at the C-terminus of the amino acid chain. In the invention, any other fluorescent polypeptide is also suitable for coupling to the protein or polypeptide.

Furthermore, in another embodiment of the invention, the marker molecule may also be a non-fluorescent marker molecule. Non-fluorescent marker molecules are all marker molecules which do not produce a fluorescent label but produce a colored reaction on the test medium caused by a chemical reaction. These are usually detected by light microscopy. In particular, peroxidases, NBT (nitro blue tetrazolium chloride), which reacts with BCIP (5-bromo-4-chloro-3-indoxyl phosphate) in color, or DAB (diaminobenzidine) can be mentioned here. Any other histochemical dye is also suitable as a non-fluorescent marker molecule. The non-fluorescent marker molecule can be present, for example, in the form of a fusion protein together with the protein or polypeptide according to the invention.

In a further embodiment of the invention, the protein or polypeptide according to the invention is coupled to a medicinal active substance. According to the invention, this can be coupled directly to the protein or polypeptide or linked to the protein or polypeptide according to the invention via another molecule, for example a so-called linker. A marker molecule in a particular embodiment of the invention may be coupled to the protein or polypeptide in addition to the medicinal agent. Likewise, it is possible that the additional marker molecule is not attached to the Protein or polypeptide itself, but is coupled to the medicinal agent which is linked to the protein or polypeptide.

The invention particularly relates to the use of a protein or polypeptide according to any one of claims 1 to 14 as a biomarker for the detection of tubulin and / or Mikrotubulistrukturen. The protein or polypeptide of the invention may be used to produce a biomarker. This can be marked inexpensively directly with a marker molecule. Thus, the biomarker can be used directly and quickly for the representation of microtubule structures. This is important not only in basic research for the further investigation of the cytoskeleton and its associated proteins, but in particular also in the diagnosis of unusual microtubule structures due to tumor formation or other lesions of cells. With the protein or peptide according to the invention is a cost-effective biomarker, which ensures a specific labeling of tubulin. The biomarker can be advantageously used both in living and in fixed cells for the representation of microtubule structures. Purified microtubule structures can also be detected independently of a cellular environment. It is also suitable according to the invention for labeling the microtubule building block tubulin. Thus, the biomarker also found use in cell lysates, ELISA assays or membrane-based experiments.

The protein or polypeptide according to the invention can also be used as part of a microtransporter for introducing active substances into cells. In this case, the shuttle function of the protein or polypeptide can be used. By virtue of its binding property to tubulin, medicinal or other active substances can be purposefully transported to their site of action, for example the microtubules of a cell. The protein or polypeptide can thus also be used for therapeutic purposes, for example in cancer therapy.

The invention also provides a method for producing a biomarker for the detection of tubulin and / or microtubule structures. According to the invention, a construct is first prepared for this purpose, which comprises a corresponding nucleic acid encoding a protein or polypeptide according to one of claims 1 to 6. In addition, the construct comprises a nucleic acid encoding a marker molecule, as well as any other factors needed for the correct expression of the biomarker, such as promoters or enhancers. For expression of the construct, this is transfected into suitable recipient cells. In the context of the invention, suitable common receptor cells are all common cloning cell lines, in particular cells of the bacterium Escherichia coli, but cells of fungal origin or algal cells are also suitable for cloning. After selection of successfully transformed cells and their multiplication, the biomarker comprising the protein or polypeptide according to the invention and one from the recipient cells is purified. The biomarker is then, for example, in the form of a fusion protein, which can be used for the direct detection of tubulin.

The construct according to the invention may additionally comprise a nucleic acid encoding a protein tag. In the context of the invention, any protein tag which allows the purification of the protein or polypeptide according to the invention is suitable. In particular, the protein tag glutathione-S-transferase (GST) should be mentioned here. Advantageously, GST promotes dimerization of its fused proteins. The invention also relates to a method for the detection of tubulin and / or microtubule structures. The protein or polypeptide according to the invention coupled to a marker molecule is added in a first step added to live cells, fixed cells or cell fractions. In the case of intact cells, it is assumed that the membrane of the cells is permeable to, or rendered permeable to, the protein or polypeptide of the invention. Subsequently, a hybridization of the biomarker according to the invention to tubulin is carried out. It is irrelevant whether the tubulin is present as a component of Mikrotubulistrukturen or as protein freely in a solution or bound to a membrane. The hybridized protein or polypeptide is detected after appropriate washing steps depending on the desired stringency. The detection can take place in the form of a visualization. The detection is preferably carried out microscopically with light sources adapted to the marker molecule.

The invention also relates to a kit for the detection of tubulin and / or microtubule structures comprising a protein or polypeptide according to any one of claims 1 to 14. The kit comprises the protein or polypeptide according to the invention, which is already connected to a marker molecule in the form of a fusion protein. Alternatively, the protein or polypeptide is provided separately to a marker. The kit comprises these components according to the invention as ready-to-use solutions or in concentrated form. Also, the kit includes an instruction for the user. Optionally, further solutions, for example buffer solutions are contained in the kit according to the invention. Furthermore, the invention relates to a recombinant eukaryotic

A cell transfected or stably transformed with a construct comprising at least one nucleic acid encoding the protein or polypeptide of any one of claims 1 to 6. Correct expression of the construct is ensured by appropriate promoters or other additional factors. The transfected into the recombinant eukaryotic cell or stably transformed construct comprises according to a Embodiment of the invention also a nucleic acid encoding a marker molecule, for example GFP or YFP, which is operatively linked to the nucleic acid coding for the protein or polypeptide according to the invention.

A cell-based screening system for medicinal agents is also an object of the invention. The screening system can be present for example in the form of a kit and according to the invention comprises the above-described recombinant eukaryotic cells.

The invention also provides a screening method for medicinal active ingredients in which the described recombinant eukaryotic cells are used. In this case, recombinant eukaryotic cells comprising at least one construct with a nucleic acid encoding a protein or polypeptide according to the invention according to one of claims 1 to 6 are provided. After adding at least one medicinal agent to the recombinant eukaryotic cells, the cells react with it. Optionally, the cells must be made permeable to the active ingredient accordingly. The influence of the active ingredient on the microtubule structures present in the cell or free tubulin is subsequently evaluated. Preferably, the evaluation is carried out microscopically. However, for example, sorting for living and dead cells (e.g., by FACS - fluorescent activated cell sorting) is also possible.

example 1

The construct GST-PEX14 (1-78) and fusion proteins with fluorescent proteins (eg GST-eYFP-PEX14 (1-78) amino acids 1 to 78 of the human peroxisomal biogenesis factor PEX14 coupled to glutathione-S-transferase and the fluorescence marker enhanced Yellow Fluorescent Protein) were cloned into pGEX4T1 vectors. Expression was overnight at 20 ° C with 1 mM IPTG. The GST fusion proteins were purified by Glutathione-Sepharose 4B (GE Healthcare) according to the manufacturer's instructions.

For fluorescence microscopy, a human fibroblast cell line, an astrocyte cell line (rat), primary neuronal cultures of the rat cerebellum and the dorsal root ganglia of the chicken, and individual trypanosomes were used:

Human fibroblasts:

GM5756T (SV-40 transformed GM5756, Corial Cell Repository, Camden NJ)

Medium:

DMEM High Glucose (PAA)

+ 10% FCS (Gibco)

+ Pen / Strep (Gibco)

+ 2mM L-glutamine (PAA)

Culture Conditions

37 ° C; 8.5% C0 ₂

Roll cultures Grain cells or purkinic cells: Sagital sections (400 pm) of rats (postnatal day 10) were used as

Cultivated roller cultures for 16 days and fixed with 4% formaldehyde. Due to the greater layer thickness compared to the monolayer cultures, all incubation times of the staining protocol were doubled.

(Meiler, Krah, Theiss, 2005, Development Brain Research 160: 101-105) Astrocvts: cell line isolated from rat cortex.

(Theiss & Melier, 2002, Cell & Tissue Research 310: 143-154)

Chicken DRG (dorsal root ganglion, spinal ganglion)

Spinal ganglia neurons explanted from chicken (embryonic day 10) were cultured for 3 days and fixed with 4% formaldehyde.

(Theiss & Melier, 2001, Journal of Neurocytology 30: 59-71)

All solutions for the detection of microtubule structures of the above mentioned Cells were seeded in D'PBS (Dulbecco's phosphate buffered saline). All steps were carried out at room temperature (RT).

Unless otherwise stated, the cells were fixed with 3% formaldehyde for 20 min or 2% glutaraldehyde for 30 min, then solubilized with 1% Triton-X100 for 5 min, whereupon nonspecific attachment sites with a 3% BSA solution ( bovine serum albumin) were blocked. Between these incubation steps, three times each was washed with D'PBS. Between the following incubation steps, each was washed three times for 5 minutes with 0.1% BSA in D'PBS. The incubations with the biomarker (10-1000 ng / μΙ) in 0.1% BSA were carried out for 30 min. For markers without fluorescence labeling, a further incubation was carried out with a monoclonal anti-GST primary and a fluorescently labeled secondary antibody (Alexa 488 or Alexa 594). The cells were then washed three times for 5 minutes with 0.1% BSA in D'PBS and twice with D'PBS and capped with slides on Mowiol embedding medium. To further verify the correct labeling of microtubules, double-labeling was also performed with the recombinant protein described above as well as commercial specific tubulin antibodies. The protocol corresponds to the one presented, in which the microtubules were displayed immunohistochemically using the two-step method (primary and secondary antibodies).

The following protocol describes the individual steps of the microtubule detection method:

1. Wash cells 3x with D'PBS (Gibco)

2. fixation with 4% formaldehyde 4% glutaraldehyde for 20 min

3. Wash 2x with D'PBS

4. Permeabilization of cell membrane with 1% Triton-X100 for 5 min

5. Wash 2x with D'PBS

6. Block unspecific binding with 3% BSA

7. Incubation with the new tubulin marker (0.2 μg / μΙ) or

First antibody (1: 200) in 0.1% BSA for 30 min

8. 3x for 5 min each wash with 0.1% BSA

9. Incubation with second antibody (1: 200) for 30 min

10.3x for 5 min each wash with 0.1% BSA

11.2x wash with D'PBS

12. Mount coverslips on microscope slides (Mowiol)

The use of the biomarker GST-eYFP-PEX14 (1-78) according to the invention eliminates the steps 8 and 9.

When using GST-PEX14 (1-78), ie without fluorescent fusion, a total of three incubation steps (GST-PEX14N, anti-GST first antibody, fluorescently labeled second antibody) are performed according to steps 7 and 8 or 9 and 10 respectively. When fixed with methanol, instead of step 2 for 5 minutes with -20 ° C cold methanol fixative reagent (90% (v / v) methanol, 10 mM MES pH6.9, 0.1 mM EGTA, 0.1 mM MgCl ₂ ) is incubated, permeabilization of the cell membrane (steps 3 and 4) is not necessary.

Unless otherwise stated, it was always fixed with formaldehyde and labeled according to the above protocol. However, in the staining of the roll cultures (granule cells or Purkinje cells) all incubation times were doubled.

For microscopic detection of microtubule structures, a Zeiss LSM 510 meta confocal laser scanning microscope was used. For enlargement the objectives Plan-Apochromat 63x / 1.4 Oil, Plan-Neofluar 40x / 1.3 Oil and Plan-Neofluar 10x / 0.3 were used. The fluorescence label eYFP (enhanced Yellow Fluorescent Protein) was detected at the wavelengths 514 nm (excitation) and 530-600 nm (emission). For the secondary antibody Alexa 594 (red), the wavelengths 543 nm (excitation) and 585 nm (emission) were used. For fluorescein, the wavelengths 488 nm (excitation) and 505-550 nm (emission) were used.

In summary, for the detection methods presented above, the following should be stated:

The biomarker PEX14 (1-78) according to the invention was expressed recombinantly in E. coli in the form of GST fusion proteins and purified by means of the GST tag. For fluorescent labeling, either an eYFP fusion portion was expressed (GST-eYFP-PEX14 (1-78)), or the purified GST-PEX14 (1-78) was specifically labeled with 5-iodoaceto on the exposed cysteine residues of the GST fusion portion - Fluorescein (5-IAF) labeled. The cells tested were treated with a standard protocol for immunofluorescence using the biomarker of the invention as an antibody, but without incubation with a second antibody, since the biomarker was already fluorescently labeled.

In all cells tested microtubules could be labeled with a very good quality using the biomarker according to the invention. In all cases, the human PEX14 (1-78) was used. The marker can therefore be used cross-species, which is particularly worth mentioning in the evolutionary far from human trypanosomes. In addition, it has been shown that the marker is equally useful as both a fluorescent fusion protein (GST-eYFP-PEX14 (1-78)) and a chemical fluorescent label (5-IAF).

In order to prove that the labeled structures are indeed microtubules, in some cases the microtubules were additionally labeled with anti-tubulin antibody. The double labels show a nearly identical labeling of microtubules with the new marker and with the antibodies.

Example 2 For more accurate characterization of the binding regions of

PEX14 (1-78) on tubulin was subjected to a so-called peptide scan. Peptide scans are used to visualize specific protein-protein interactions. For localization of specific binding sites every second

15mer peptide from PEX14 (1-78) synthesized and fixed on a cellulose membrane. Control experiments showed that the used Antibodies did not cross-react with the synthetic peptides (data not shown). The 15-mer peptides fixed on the membrane were then incubated with 1 nM of pure tubulin and hybridized with anti-tubulin antibodies.

As shown in Figure 9, some of the 15-mer peptides hybridized with tubulin, giving an indication of the binding range of PEX14 (1-78) to tubulin. The peptide scan shows that tubulin does not bind in the sequence region of amino acids 1-33 and 58-78. However, a high binding affinity for tubulin was found particularly in the peptides containing lysine at position 34 of PEX14 (1-78). Likewise, good binding to tubulin was found in peptides containing phenylalanine at position 52 of PEX14 (1-78). The intensity of binding was particularly strong in peptides containing phenylalanine at position 52 and lysine residues at positions 54, 55 and 56 of PEX14 (1-78). The intensity of hybridization of the 15-mer peptides to tubulin decreased as soon as alanine, threonine and arginine were present in close proximity to lysine (34) and phenylalanine (52). This indicates that alanine, threonine and arginine may also play an important role in the binding of PEX14 (1-78) to tubulin, possibly being necessary for correct folding of the peptide or for free access of lysine to tubulin.

Exemplary embodiments of the protein or polypeptide according to the invention and its use as a biomarker are additionally illustrated by the following FIGS. 1 to 8:

FIG. 1 shows the three-dimensional structure of the protein or polypeptide according to the invention based on the amino acids 20 to 78 derived from the human peroxisomal biogenesis factor PEX14. As can be seen from the illustration, three α-helices are U-shaped arranged to each other. A fourth diagonal helix stabilizes the U-shaped structure.

Fig. 2 shows cells from the spinal ganglion of the chicken (Gallus gallus). Using the biomarker construct GST-eYFP-PEX14 (1-78) according to the invention (amino acids 1 to 78 of the human peroxisomal biogenesis factor PEX14 coupled to glutathione S-transferase and the fluorescence marker-enhanced yellow fluorescent protein), microtubule structures were fluorescence-stained in these cells. On the right image a growth cone with high resolution is visible.

Fig. 3 shows the staining of microtubule structures in chicken fibroblasts (Gallus gallus) by hybridization of the biomarker construct GST-eYFP-PEX14 (1-78) to tubulin.

FIG. 4 shows the staining of microtubule structures on rat astrocytes (Rattus rattus) by hybridization of the biomarker construct GST-eYFP-PEX14 (1-78) to tubulin. Fig. 5 shows the labeling of microtubules in human fibroblasts

(GM5756) with GST-PEX14N, which after the purification with 5-IAF (5-iodoaceto-fluorescein, Sigma-Aldrich) according to manufacturer's instructions on cysteines was labeled. Fig. 6 shows microtubules in human fibroblasts (GM5756), which with

GST-eYFP-PEX14 (1-78).

Figure 7 shows microtubules in a granule preparation (rat) labeled with GST-eYFP-PEX14 (1-78).

Fig. 8 shows microtubules in trypanosomes (7 ^" brucei) simultaneously with GST-eYFP-PEX 4 (1-78) and with anti-tubulin antibodies, and a red secondary antibody. The left column shows the eYFP label, the middle column shows the label with anti-tubulin antibodies and red secondary antibody. The right column shows the superimposition of the first and second detection methods.

Figure 9 shows the result of hybridization of 1 nM pure tubulin to a membrane populated with 15-mer peptides from PEX14 (1-78). The detection was carried out with anti-tubulin antibodies (1: 3000). On the right side, the amino acid sequences of the 15-mer peptides are shown schematically. A strong hybridization of tubulin is particularly evident in the 15-mer peptides A3, B3, C3, D3 and A4 - these contain lysine at the original position 34 of PEX14 (1-78). Strong binding to tubulin is also evident in the 15-mer peptides E4, A5, B5, C5, D5 and E5 - these contain phenylalanine at the original position 52 of PEX14 (1-78) and also 3 lysine residues at their original positions 54, 55 and 56.

Claims

A protein or polypeptide comprising an amino acid sequence comprising at least one lysine-rich sequence region, wherein the protein or polypeptide binds to tubulin.

2. Protein or polypeptide according to claim 1, characterized in that the amino acid sequence at least the sequence

Phe-Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa Lys.

3. Protein or polypeptide according to claim 1 or 2, characterized in that the amino acid sequence at least the sequence

Lys-Phe-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Phe-Xaa-Xaa-Xaa-Lys (SEQ ID NO: 1) 1).

4. Protein or polypeptide according to one of the preceding claims, characterized in that the amino acid sequence at least the sequence

Lys-Phe-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Phe-Xaa-Lys-Lys-Lys (SEQ ID NO: 1) 2).

5. A protein or polypeptide according to any one of the preceding claims comprising an amino acid sequence selected from the group comprising

a) an amino acid sequence according to SEQ ID NO: 3 or a

Partial sequence thereof; or

b) a sequence derived from SEQ ID NO: 3 by substitution, insertion or deletion of amino acids.

6. Protein or polypeptide according to one of the preceding

Claims comprising an amino acid sequence according to SEQ ID NO: 4.

7. Protein or polypeptide according to one of the preceding claims, characterized in that the protein or polypeptide forms a three-dimensional structure which comprises at least three a-helices.

8. Protein or polypeptide according to one of the preceding claims, characterized in that the protein or polypeptide is present as a dimer in its binding to tubulin.

9. Protein or polypeptide according to one of the preceding claims, characterized in that the protein or polypeptide is coupled to at least one heterologous molecule.

10. Protein or polypeptide according to claim 9, characterized in that the heterologous molecule is a marker molecule.

11. Protein or polypeptide according to claim 10, characterized in that the marker molecule is a fluorescent marker molecule.

12. Protein or polypeptide according to claim 11, characterized in that the fluorescent marker molecule is a polypeptide.

13. A protein or polypeptide according to claim 12, characterized in that the polypeptide is a fluorescent polypeptide selected from the group comprising GFP, YFP, CFP, BFP, drFP583 or another fluorescent polypeptide.

14. A protein or polypeptide according to claim 10, characterized in that the marker molecule is a non-fluorescent marker molecule selected from the group comprising peroxidases, NBT-BCIP, DAB or another non-fluorescent marker molecule.

15. A protein or polypeptide according to claim 10, characterized in that the heterologous molecule is a medicinal agent which is directly or indirectly coupled to the protein or polypeptide.

16. A protein or polypeptide according to claim 15, characterized in that the protein or polypeptide is additionally coupled to a marker molecule.

17. Use of a protein or polypeptide according to any one of claims 1 to 14 as a biomarker for the detection of tubulin and / or

Mikrotubulistrukturen.

18. Use of a protein or polypeptide according to claim 17 in living cells or fixed cells or cell fractions or membrane-based assays.

19. Use of a protein or polypeptide according to claim 15 as a component of a microtransporter for introducing active substances into cells.

20. A method for producing a biomarker for the detection of tubulin and / or microtubule structures comprising the following steps:

a) cloning a construct comprising a nucleic acid encoding a protein or polypeptide according to any one of claims 1 to 6 and coding for a marker molecule

Nucleic acid into a vector;

b) transfecting the vector into a recipient cell, especially in E. coli;

c) increasing the transfected cells;

d) Purification of the biomarker from the recipient cells.

21. The method according to claim 20, characterized in that the construct comprises a nucleic acid encoding a protein tag, in particular a nucleic acid encoding the protein-tag glutathione-S-transferase, with the nucleic acid encoding the protein or polypeptide according to a of claims 1 to 6 is operatively connected.

22. A method for the detection of tubulin and / or microtubule structures comprising the following steps:

a) adding a protein or polypeptide according to any one of claims 9 to 14 to living cells or fixed cells or cell fractions;

b) carrying out a hybridization of the protein or polypeptide according to any one of claims 9 to 14 to tubulin;

c) detecting the hybridized protein or polypeptide.

23. Kit for the detection of tubulin and / or microtubule structures comprising a protein or polypeptide according to one of claims 1 to 14.

24. Recombinant eukaryotic cell, characterized in that the cell is transfected or stably transformed with a construct which comprises at least one nucleic acid coding for the protein or polypeptide according to one of claims 1 to 6.

25. Recombinant eukaryotic cell according to claim 24, characterized in that the construct comprises a coding for a marker molecule nucleic acid operatively linked to the nucleic acid encoding the protein or polypeptide according to claim 1 to 6.

26. A cell-based medical drug screening system comprising recombinant eukaryotic cells according to claim 24 or 25.

27. A method of screening medical drugs comprising the steps of:

a) providing recombinant eukaryotic cells according to claim 24 or 25;

b) adding at least one medicinal agent;

c) Evaluation of the reaction result.