EP3201328A1

EP3201328A1 - Fusion protein and purifying method

Info

Publication number: EP3201328A1
Application number: EP15771867.7A
Authority: EP
Inventors: Johann Kubicek; Frank Schäfer; Jörg LABAHN
Original assignee: Individual
Current assignee: Individual
Priority date: 2014-09-05
Filing date: 2015-09-07
Publication date: 2017-08-09
Also published as: WO2016034741A1; DE102014217793A1; US20170260255A1

Abstract

The invention relates to, inter alia, a fusion protein comprising a protein of interest and an affinity tag which binds lysozyme. Lysozyme can be used to purify, precipitate, or support the crystallization of such a fusion protein. The invention further relates to a binding agent which specifically binds a target compound. The binding agent has a CDR3 region which is derived from a single-domain antibody but which does not have framework regions or other elements for stabilizing the CDR3 region, and the binding agent binds the target compound via the CDR3 region.

Description

"Fusion protein and purification process"

FIELD OF THE INVENTION

The present invention relates to novel binding agents capable of binding a target compound. The binding agents are particularly useful for therapy and as detection reagents for the detection of a target compound, for example in assays for diagnostic purposes. The binding agents can also be used as an affinity tag.

Further, the present invention relates to a capture system comprising an affinity tag and an affinity ligand useful for purifying a protein of interest. Furthermore, specific uses of the corresponding system are described, in particular for the crystallization or purification of a protein of interest.

BACKGROUND OF THE INVENTION

There is a continuing need in the art for the provision of novel binding agents which have binding affinity for a target compound. In particular, there is a need for small binding compounds which can be prepared synthetically and / or recombinantly in a simple manner and which, despite their small size, can specifically bind to a target compound. Corresponding small binding molecules also offer the advantage that they may be able to penetrate barriers in the body, such as. As the blood-brain barrier, which can not be penetrated by conventional binding agents (for example, antibodies) due to their size. Corresponding small binding agents would therefore have considerable advantages in therapeutic and diagnostic fields. Thus, it is an object of the present invention to provide compounds which are capable of specifically binding to a target compound but which are smaller than, for example, antibodies or commonly used antibody fragments. Recombinant DNA technology enables the production of desired proteins of interest in host cells. Such polypeptides produced by host cells are typically separated from the proteins of the host cell prior to use to obtain them in pure form. Affinity chromatography based methods are often preferred for protein purification. Affinity chromatography can be used to purify high-yield proteins from complex mixtures. Affinity chromatography is based on the ability of proteins to bind noncovalently but nevertheless specifically, for example to a ligand immobilized on a support for the desired protein, for example to an antibody which binds the protein to be purified. If the protein of interest has affinity for a metal ion, isolation can be accomplished using metal affinity chromatography, as is the case with IMAC methods, for example.

Furthermore, a protein of interest is often purified by expressing the protein of interest as a fusion protein, wherein the fusion protein comprises the protein of interest and one or more affinity tags. The affinity tag allows for the purification of the protein of interest according to generalized protocols as opposed to the highly specific procedures associated with conventional chromatography. These tags offer the great advantages of, for example, conventional metal affinity tags (for example, when using a poly-His tag), namely, the ability to inexpensively large scale purification, as well as possible cleaning under denaturing conditions, and so on.

Several such cleaning tags are known in the art, such as poly His tag, streptavidin tag, SBP tag, GST tag, and similar purification tags that allow the isolation of various proteins over the same affinity tag. Despite the numerous affinity tags that are already available, there remains a need in the art for improved purification methods, particularly methods that permit purification of the protein of interest using an affinity ligand that can either be attached to a solid support or which allows purification of the protein of interest without the use of a solid support.

Thus, it is further an object of the present invention to provide a method for purifying a protein of interest.

Further, an object of the present invention is to provide means for assisting the crystallization of a protein of interest. SUMMARY OF THE INVENTION

One aspect of the present invention is based on the surprising discovery that the isolated CDR3 region of heavy chain antibodies, in particular the CDR3 region derived from camelid antibodies (also called the CDR3 region), binds as a binding agent Binding of a target compound can be used even if the stabilizing frameworks of the heavy chain antibody (and / or other commonly used stabilizing structures) are removed or not used. This finding is very surprising since it has been previously believed that the stabilizing frameworks or similar stabilizing agents are imperative to give the CD3 region the correct conformation or 3D structure to bind the CDR3 region to its target compound enable. However, it has now been found that the CDR3 region alone, without conformation stabilizing structures, is capable of binding to a target compound with substantially the same specificity and / or affinity as the heavy chain antibody from which the CDR3 region is derived tie. This discovery allows the provision of binding agents that are even smaller than conventional heavy chain antibodies, but still bind their target compound with the desired specificity and / or affinity.

Thus, in one aspect, the present invention provides a binding agent that specifically binds to a target compound, wherein the binding agent has a CDR3 region derived from a heavy chain antibody, but no framework regions for stabilizing the CDR3 region or other stabilizing Having structures for stabilizing the CDR3 region and wherein the binding agent is the

Target compound binds across the CDR3 region. Further provided is a pharmaceutical composition having a corresponding binding agent.

Another essential aspect of the present invention is based on the idea of using a specific affinity tag-based system that makes it possible to purify, precipitate and / or crystallize a protein of interest. This system is based on the use of lysozyme as the affinity ligand and the use of a lysozyme binding affinity tag. The inventors have found that this specific combination of an affinity tag that binds lysozyme and lysozyme as an affinity ligand offers significant advantages in the purification, precipitation, and / or crystallization of a protein of interest.

Further provided is a fusion protein comprising a protein of interest and an affinity tag that specifically binds lysozyme. Preferably, the fusion protein is produced recombinantly. Further provided is a method for purifying a fusion protein comprising a protein of interest and an affinity tag specifically binding lysozyme, the method comprising a step of employing lysozyme as the affinity ligand attached to the affinity tag of the protein Fusion protein binds. This forms a complex comprising lysozyme and the fusion protein to be purified from the sample. The complex can then be separated from the remainder of the sample and the fusion protein released from the complex. Lysozyme may be added in free form to the sample containing the fusion protein, or immobilized on a support, such as a column.

Thus, the present invention also relates to a complex having lysozyme as affinity ligands, wherein the lysozyme is bound to a fusion protein having a protein of interest and a lysozyme binding affinity tag. Further, the present invention relates to an expression vector for expression of a fusion protein, wherein the fusion protein comprises a protein of interest and an affinity tag specifically binding lysozyme. Furthermore, a host cell is provided which has a corresponding expression vector. Further, the present invention relates to the use of lysozyme to assist and / or facilitate the crystallization of a fusion protein having a protein of interest and an affinity tag that binds lysozyme.

Further, the present invention relates to the use of lysozyme for the purification of a fusion protein comprising a protein of interest and an affinity tag that binds lysozyme.

Further, the present invention relates to the use of lysozyme for the precipitation of a fusion protein having a protein of interest and an affinity tag that binds lysozyme.

Other objects, features, advantages and aspects of the present application will become apparent to those skilled in the art from the following description and the appended claims. It is to be noted, however, that the following description, the appended claims and the specific examples, when indicating preferred embodiments of the application, are given by way of illustration only. Those skilled in the art will, after reading the following, be susceptible to various changes and modifications in the scope and spirit of the disclosed invention. DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered that, unlike prior art views, it is possible to use the CDR3 region derived from a heavy chain antibody without its framework regions or other conformation stabilizing structures as a binding agent to bind a target compound.

Thus, in accordance with one aspect of the present invention, there is provided a binding agent that specifically binds a target compound, wherein the binding agent has a CDR3 region derived from a heavy chain antibody, but does not have the corresponding framework regions or other structures containing the Stabilize CDR3 region, and wherein the binding agent binds the target compound across the CDR3 region. The CDR3 region according to the invention is derived from a heavy chain antibody. The CDR3 region is preferably derived from a heavy chain antibody of camelids (such as dromedaries, camels or alpacas), but may also be derived from a heavy chain antibody from other animals that produce corresponding antibodies. such as B. sharks. A CDR3 region is \ "derived \" from such a heavy chain antibody when the CDR3 region has homology or identity with the corresponding CDR3 region of the reference heavy chain antibody of at least 80%, at least over its entire length 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99% or 100% The CDR3 region specific for the target compound can be obtained, for example, from the above-mentioned antibodies by means of which appropriate

Heavy chain antibodies to a target compound can be obtained by those skilled in the art, for example, by using immunization methods known in the art. For example, the dromedary, camel or shark may be immunized with the desired target compound as an antigen and the mRNA encoding heavy chain antibodies subsequently isolated. By reverse transcription and

Polymerase chain reaction can be a gene library of single domain antibodies containing several million clones generated. Also screening methods, such as. Phage display and ribosome display, can be used to identify the antigen-binding clones. The present invention further encompasses obtaining suitable CDR3 binding regions from heavy chain antibody libraries by screening methods. If a suitable CDR3 region is identified, it can also be manufactured synthetically thanks to its short length. This represents a significant advantage over prior art binding agents which, because of their size, usually need to be prepared recombinantly. According to one embodiment, the CDR3 region has a length of 10 to 30, preferably 12 to 25 amino acids. The CDR3 region of such heavy chain antibodies has the unusual ability to form finger-like extensions that can protrude into cavities of antigens, for example, into the groove of the active site of an enzyme. For example, the CDR3 region may form convexities or protrusions that may occupy, for example, the groove of a target compound. Thus, in one embodiment, the CDR3 region attaches to a groove, pocket or canyon of a target compound. The non-stabilized CDR3 region of the present invention penetrates into the groove, pocket or canyon of the target compound and is thus stabilized by the molecular interactions between the CDR3 region and the lining amino acids of the groove, pocket or canyon. In one embodiment, the CDR3 region may mimic the binding of the natural substrate to the pocket, groove or canyon of the target compound to which the CDR3 region binds.

According to one embodiment, the CDR3 region is not limited by intramolecular bonds, e.g. Disulfide bonds, cyclized. In one embodiment, the CDR3 region does not contain amino- and / or carboxy-terminal amino acid residues that would allow cyclization and thus stabilization of the CDR3 region. In one embodiment, the CDR3 region does not contain cysteine residues. Thus, the CDR3 region according to this embodiment has no cysteine residue. A cysteine residue in the CDR3 region increases the risk of nonspecific reactions across the cysteine residue (for example, by forming disulfide bonds). Therefore, it is preferred to remove or replace any cysteine residues present in the CDR3 region used for binding. For example, a cysteine residue present in the CDR3 region may be replaced with other amino acids, e.g. by serine or alanine or other suitable amino acids. Preferably, an amino acid is selected for substitution, which maintains or even improves the binding ability. In one embodiment, the binding agent is the CDR3 region. Of the

However, the scope of the present invention encompasses conjugating the CDR3 region to other compounds and / or structures, for example compounds that extend the half-life of the CDR3 region, such as HSA, HES or PEG, marker compounds or cytotoxic agents. Also, attachment to other protein or peptide compounds is possible, wherein, for example, the CDR3 region is conjugated or fused via a synthetic linker or via a glycine or alanine linker or by other linking agents to form a fusion construct. The conjugation and / or binding may be covalent or noncovalent. However, according to one embodiment, the conjugation does not stabilize the conformation of the CDR3 region used as the binding agent. According to a preferred embodiment, the binding agent binds via the CDR3 region with the same specificity and / or affinity to its target compound as the heavy chain antibody from which it is derived and which has the corresponding framework regions. As discussed above, the inventors have surprisingly found that the CDR3 region of heavy chain antibodies used to bind the target compound according to the present invention retains its binding specificity despite the fact that the conformation of the CDR3 region does not the framework regions of the heavy chain antibody or other stabilizing structures is stabilized. This has the advantage that the binding agent can be made small and its production is also considerably simplified since it can be produced synthetically and it is not necessary, for example, to cyclize the CDR3 region or to add conformation-stabilizing structures.

In one embodiment, the binding agent binds lysozyme as the target compound across the CDR3 region. As stated, the binding agent may also consist of the CDR3 region.

For binding lysozyme, in one embodiment, the binding agent comprises a CDR3 region having or consisting of a sequence selected from the group consisting of

SEQ ID NO 1: DSTIYASYYECGHGLSTGGYGYDS

SEQ ID NO 2: DTSTWYRG YCGTN P N YFS Y

SEQ ID NO 3: GWSSLGSCGTNRNRYNY

SEQ ID NO 4: GYRNYGQCATRY

SEQ ID NO 5: GYRNYGQSATRY

SEQ ID NO 6: TRKYVPVRFALDQSSYDY

The examples of this application show that the above isolated CDR3 regions of heavy chain antibodies can bind lysozyme and therefore can be used as binding agents for lysozyme. These binding agents, which as stated can also consist of one of the above sequences, can advantageously also be used as an affinity tag which has an affinity for lysozyme. This is described in detail below.

Further, the present invention provides a pharmaceutical or diagnostic composition comprising a binding agent according to the present invention. Due to the small size of the binding agent, a corresponding pharmaceutical or diagnostic composition has several advantages. First, their easy and safe preparation is advantageous for therapeutic and diagnostic uses. Furthermore, the small size of the binding agent allows for example Penetration of barriers in the body, such as the blood-brain barrier, which can not be penetrated by larger molecules, such as antibodies. The binding agent according to the present invention can be used analogously to conventional antibodies and binding agents in therapy and diagnosis. For example, the binding agent can be used to detect a target compound so as to enable diagnosis based on the presence or absence of the target compound.

Further, the binding agent according to the present invention can be advantageously used as an affinity tag that allows easy isolation of a protein of interest when the affinity tag is fused to the protein of interest. Such a recombinant fusion can be achieved, for example, by expressing the protein of interest and the affinity tag as a fusion construct. Thus, in one embodiment, the present invention provides a fusion protein having a protein of interest and an affinity tag, wherein the affinity tag has or consists of a CDR3 region derived from a heavy chain antibody however, contains no framework regions for stabilizing the CDR3 region. Preferably, the CDR3 region also has no other stabilizing structures. As discussed above, the CDR3 regions derived from corresponding heavy chain antibodies have several advantages that make them particularly suitable for use as an affinity tag. They are small and surprisingly do not require stabilizing frameworks or other conformation stabilizing structures to bind with high specificity to a target compound and thus an affinity ligand. Thus, they can easily be expressed as a fusion protein together with the protein of interest. As discussed above, the CDR3 region containing the

Binding function and thus can serve as an affinity tag, preferably a length of 10 to 30, particularly preferably 12 to 25 amino acids on. In one embodiment, the CDR3 region forms a finger-like spur or protrusion that can protrude into the cavity, groove, or pocket of an affinity ligand, for example, into the groove of the active site of an enzyme. Thus, the CDR3 binds

A region according to one embodiment of a groove, a pocket or a canyon of a target compound, which is used as an affinity ligand. Binding is achieved by, for example, penetrating the CDR3 region into the groove, pocket or canyon of the target compound and being stabilized by the molecular interactions between the CDR3 region and the amino acids lining the groove, pocket or canyon , The affinity ligand therefore preferably has a corresponding groove, pocket or canyon and is most preferably an enzyme such as lysozyme. This preferred embodiment will be described in more detail below. The affinity tag is preferably unrelated to the protein of interest and therefore, of course, is not expressed as a corresponding fusion protein.

The recombinant fusion protein can then be isolated via the affinity tag CDR3 region, which specifically binds to the corresponding affinity ligand. For example, the affinity ligand may be immobilized on a solid support such as a column, or it may be added directly in free form to a fusion protein-containing mixture to precipitate the fusion protein, as described below for the preferred example of lysozyme as the affinity ligand.

In another aspect, the present invention relates to a novel affinity tag / affinity ligand system based on the use of lysozyme as an affinity ligand and a lysozyme-specific affinity tag. This novel affinity tag / affinity ligand system, based on the use of lysozyme as the affinity ligand, has several advantages and wide applications in the fields of protein purification, protein precipitation and / or protein crystallization.

In another aspect, there is provided a fusion protein comprising a protein of interest and an affinity tag capable of binding lysozyme. Such a fusion protein, which can be produced, for example, recombinantly, can easily be produced by the

Methods according to the present invention are purified using lysozyme as the affinity ligand. The lysozyme-binding affinity tag is not naturally associated with the protein of interest, but the fusion construct is generated, for example, by recombinant DNA technology.

The fusion protein according to this aspect of the present invention has a specific affinity tag that binds to lysozyme, thereby allowing specific, affinity-based isolation of the fusion protein. In one embodiment, the lysozyme-binding affinity tag is located at either the N-terminus or the C-terminus of the fusion protein. Preferably, the affinity tag is according to the present invention

Invention arranged at the C-terminus. However, the location of the affinity tag may depend on the fusion protein to be expressed and its intended use. One of the advantages of using an N-terminal affinity tag is that the yield of the expressed fusion protein is increased, providing a reliable context for efficient translation. The use of an N-terminal

However, affinity tags do not always result in the purification of full-length proteins because defective translation products that do not contain the protein of interest in full length are also scavenged due to the N-terminal affinity tag. Therefore, for most applications, a C-terminus affinity tag will be advantageous because purification of the full-length fusion protein is enhanced compared to N-terminal affinity tag fusions. In a preferred embodiment, the affinity tag binds to the active site of lysozyme. The inventors have found that it is advantageous if the affinity tag binds to the active site of lysozyme, as this gives high specificity to the affinity ligand lysozyme. Furthermore, the binding of the affinity tag to the active site of lysozyme allows the use of mild elution conditions in the purification of the fusion protein, for example when using sugars bound by lysozyme. Corresponding sugars can displace the fusion protein or the affinity tag from the active center of the lysozyme and thereby release it from the complex. Thereby, the fusion protein can be separated from the affinity ligand lysozyme as described below in connection with the purification process according to the present invention. This promotes elution.

In one embodiment, the affinity tag comprises or consists of an immunoglobulin molecule or a functional fragment thereof that binds lysozyme. In particular, the immunoglobulin molecule may be an antibody.

In particular, the term "antibody" refers to a protein having at least two heavy chains and two light chains linked by disulfide bonds The term "antibody" includes naturally occurring antibodies as well as all recombinant forms of antibodies, for example, non-glycosylated antibodies expressed in prokaryotes Antibodies, humanized antibodies and chimeric antibodies. Each heavy chain consists of a heavy chain variable range (VH) and a heavy chain constant range (CH). Each light chain consists of a light chain variable range (VL) and a light chain constant range (CL). The heavy chain constant range comprises three or, in the case of antibodies of the IgM or IgE type, four

Heavy chain constant domains (CH1, CH2, CH3 and CH4), wherein the first constant domain CH1 is adjacent to the variable region and may be linked by a hinge region to the second constant domain CH2. The light chain constant range consists only of a constant domain. The variable regions may also be divided into regions of hypervariability called complementarity determining regions (CDRs) with intermediate, more conserved regions called framework regions (FR), each variable region having three CDRs and four FRs. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g. effector cells) and the first component (C1 q) of the classical complement system.

Further, the term immunoglobulin molecule or a functional fragment thereof includes, but is not limited to, a protein or glycoprotein derived from a

Antibody is derived and capable of the same antigen, in particular the same Epitope how to bind the antibody. Thus, a fragment or derivative of an antibody as used herein generally refers to a functional fragment or derivative capable of binding to the antigen. According to a particularly preferred embodiment, the fragment or derivative of an antibody has a heavy chain variable region. It has been demonstrated that the antigen-binding function of an antibody can also be exercised by fragments of a full-length antibody or derivatives thereof. Examples of fragments or derivatives of an antibody include (i) Fab fragments, monovalent fragments consisting of the variable region and the first constant domain, each of the heavy and the light chain; (ii) F (ab) ₂ fragments, bivalent fragments comprising two Fab fragments joined at the hinge region by a disulfide bridge; (iii) Fd fragments consisting of the variable region and the first constant domain CH1 of the heavy chain; (iv) Fv fragments consisting of the heavy chain and the light chain variable region of a single arm of an antibody; (v) scFv fragments, Fv fragments consisting of a single polypeptide chain; (vi) (Fv) ₂ fragments consisting of two Fv fragments covalently linked together; (vii) a heavy chain variable domain; and (viii) multiple bodies consisting of a heavy chain variable region and a light chain variable region covalently linked such that the combination of the heavy chain and the light chain variable Area intermolecular but not intramolecular can be done. These antibody fragments and derivatives can be obtained using conventional techniques known to those skilled in the art.

Preferably, the affinity tag is derived from a heavy chain antibody. Heavy chain antibodies are well known in the art and can be obtained from camelids, for example, as described in detail above. The use of appropriate heavy chain antibodies has the advantage that they preferentially bind to the active site of the enzyme against which they were produced. Therefore, appropriate heavy chain antibodies are particularly suitable as an affinity tag for binding to the active site of lysozyme. Furthermore, the use of heavy chain

Antibodies or functional, i. Lysozyme-binding fragments have the advantage that these affinity tags are comparatively small, which is an advantage for an affinity tag, since the producing host cell need only use little capacity for the production of the affinity tag and thus the expression of the protein of interest with high yield is ensured.

According to a preferred embodiment, the affinity tag contained in the fusion protein has one or more features of the above-described binding agent having a CDR3 region of a heavy chain antibody. Thus, the affinity tag is preferably a CDR3 derived from a heavy chain antibody.

Area. The features and preferred embodiments of the CDR3 area are above described in detail and reference is made to the above disclosure, which also applies here. As discussed above, the isolated CDR3 region of corresponding heavy chain antibodies has several advantages that make it suitable for use as an affinity tag. It is small and, surprisingly, does not have to be stabilized by framework regions or other structures in order to be able to bind a target compound and thus an affinity ligand with high specificity. Thus, such a CDR3 region can easily be expressed as a fusion protein together with the protein of interest and serve as an affinity tag. As discussed above, the CDR3 region which exerts the binding function on the affinity ligands preferably has a length of 10 to 30, preferably 12 to 25 amino acids. In one embodiment, the CDR3 region forms at least one finger-like spur or protrusion which can protrude into the lumen, groove or pocket of the affinity ligand lysozyme. Thus, in one embodiment, the CDR3 region attaches to a groove, pocket or canyon of a lysozyme used as the affinity ligand. The binding is achieved by, for example, penetrating the CDR3 region into the groove, pocket or canyon of lysozyme and being stabilized by molecular interactions between the CDR3 region and the amino acids lining the groove, pocket or canyon.

In one embodiment, the affinity tag has a sequence selected from the group consisting of

SEQ ID NO 1: DSTIYASYYECGHGLSTGGYGYDS

SEQ ID NO 2: DTSTWYRG YCGTN P N YFS Y

SEQ ID NO 3: GWSSLGSCGTNRNRYNY

SEQ ID NO 4: GYRNYGQCATRY

SEQ ID NO 5: GYRNYGQSATRY

SEQ ID NO 6: TRKYVPVRFALDQSSYDY or the affinity tag consists of such a sequence. As shown in the examples, the corresponding isolated unstabilized CDR3 regions specifically bind

Lysozyme and are thus suitable for use as an affinity tag. In SEQ ID NO: 5, a cysteine was changed to a serum as compared with SEQ ID NO: 4.

In one embodiment, the fusion protein according to the present invention has a proteolytic cleavage site. Preferably, the proteolytic interface separates the

Protein of interest from the affinity tag and thus allows the removal of the affinity tag from the fusion protein, so as to obtain the protein of interest without affinity tag. Preferably, the proteolytic cleavage site is a peptide sequence that typically has about 5-30 amino acids and is a recognition motif that is an interface contains. Preferably, a proteolytic cleavage site is used which is not recognized by any protease of the host cell in which the fusion protein is expressed by means of an expression vector. This to ensure that the fusion protein is expressed with the affinity tag so as to allow for isolation of the fusion protein and not previously cleaved by the proteases of the host cell. There are several proteases that can later be used to release the affinity tag from the fusion protein so that subsequently the protein of interest can be purified without an affinity tag. Further, the present invention relates to an expression vector for expressing a fusion protein having a protein of interest and a lysozyme-binding affinity tag. As described above, according to one embodiment, the affinity tag may have a CDR3 region or consist of a CDR3 region derived from a heavy chain antibody, but not linked to the corresponding framework regions to stabilize the CDR3 region , The affinity tag can bind lysozyme. The expression vector allows the expression of a fusion protein comprising a protein of interest and a lysozyme binding affinity tag according to the present invention. The expression vector has one or more functional elements enabling expression of the fusion protein in a host cell. These functional elements may be selected from the group consisting of a promoter, an enhancer, a transcription termination signal sequence, a poly (A) site, an intron for enhancing expression of the fusion protein and a secretory signal sequence for secretion of the fusion protein. A secretory signal sequence is a peptide sequence which typically has about 10 to 30 amino acids, preferably about 15 to 30 amino acids. The secretory signal sequence is usually located at the N-terminus of the expression fusion protein and allows secretion of the fusion protein, ie, passage through a cell membrane, for example, into the cell culture or extracellular medium or into the periplasmic space. Here is the

Signal sequence is usually cleaved, so that then the fusion protein is secreted. Several suitable secretory signal sequences are known in the art which allow secretion from eukaryotic or prokaryotic cells. Suitable secretory signal sequences are described, for example, in WO 2008/000445, the disclosure of which is incorporated herein by reference.

Suitable promoters which allow the expression of fusion proteins in prokaryotic and / or eukaryotic cells are well known to those skilled in the art and therefore need not be further described. Suitable promoters include, for example, the CMV promoter, the SV40 promoter, the lacZ promoter, the ubiquitin promoter, regulatable promoters or constitutive promoters, and the like. Also, enhancer sequences for upregulating the expression of a fusion protein are known in the art. Enhancer sequences can be obtained from any eukaryotic or prokaryotic host, preferably in conjunction with the corresponding promoter that is used. Enhancer elements may include CMV enhancers or one or more glucose-dependent elements and the like. Corresponding enhancer elements are well known in the art, as well as promoter / enhancer combinations, which may also be used in accordance with the teachings of the present invention.

Furthermore, the above-described expression vector may contain further transcriptional and / or translational signals, which are preferably recognized by the corresponding host, such as e.g. B. transcriptional regulatory signals and translation initiation signals. Transcriptional and / or translational signals may be derived from any eukaryotic, prokaryotic, viral, bacterial, fungal or plant origin, preferably from human hosts or animal hosts, for example, mammalian hosts, preferably in conjunction with the corresponding promoters used. For this purpose, depending on the nature of the host cells, a wide variety of transcriptional and translational regulatory sequences may be employed. Insofar as the host cell recognizes the transcriptional regulatory signals and translation initiation signals contained in the expression vector according to the present invention, the naturally occurring 5 'regions adjacent to components of the fusion protein may also be retained in the expression vector and for translation regulation according to the present invention Invention can be used. However, other regulatory signals may also be used. The design of corresponding expression vectors is known to the person skilled in the art.

In addition to the regulatory sequences described above, the expression vector according to the present invention may include an origin of replication. Suitable origins of replication include, but are not limited to, ColE1, pSC101, SV40, ori-pMP1, and M13 origins of replication. Depending on the host cell used, the origin of replication should be active in either prokaryotic or eukaryotic host cells or both.

Suitable host cells include, for example, prokaryotic and eukaryotic host cells, for example, bacterial, fungal, plant, human and animal host cells.

Preferred prokaryotic host cells may be derived, for example, from bacteria, such as e.g. From Escherichia coli, B.subtilis, Salmonella, Pneumococcus and so on, or from algae, fungi and so on. Furthermore, eukaryotic host cells can be used, such. As animal cells or human cells, such as rodent cells such. B. CHO cells. Cells from eukaryotic organisms are particularly preferred when post-translational modifications, for example specific glycosylations, of the protein of interest are required. The host cell may also be derived from yeast. Suitable host cells are also known to the person skilled in the art.

Furthermore, the eukaryotic expression vector according to the present invention may comprise one or more selectable marker genes. Suitable selection markers are well known in the art and may, for example, be selected from the group of eukaryotic or prokaryotic selection markers. For example, the selection markers may confer resistance to antibiotics such as ampicillin, hygromycin, kanamycin, neomycin or the like. Other selection markers include, but are not limited to, for example, DHFR, GS, and the like.

Furthermore, a host cell is provided which has an expression vector according to the invention. This has been described in detail above and reference is made to the corresponding disclosure.

Further provided is a method for purifying a fusion protein comprising a protein of interest and a binding agent according to the present invention as an affinity tag, wherein an affinity ligand capable of binding the affinity tag of the fusion protein is used to purify the fusion protein. The fusion protein, and in particular the affinity tag containing it, preferably have the features described above. Reference is made to the above disclosure. As discussed above, the binding agent used as an affinity tag has a CDR3 region derived from a heavy chain antibody, but contains no framework regions, and preferably no other structures for stabilizing the CDR3 region. In one embodiment, it is used as an affinity tag

Binding agent from such a CDR3 region. The affinity ligand and the fusion protein form a complex that can be separated from the remainder of the sample to isolate the fusion protein. As stated, according to one embodiment, the affinity tag can bind lysozyme. Suitable lysozyme binding sequences have been disclosed above.

Also provided is a method of purifying a fusion protein from a sample comprising the fusion protein, wherein the fusion protein comprises a protein of interest and an affinity tag that binds lysozyme. The fusion protein and in particular the affinity tag preferably have those described above

Features on. Reference is made to the above disclosure, which also applies here.

An important feature of the method is that lysozyme is used as the affinity ligand to specifically bind the affinity tag of the fusion protein. The affinity ligand lysozyme is bound by the affinity tag of the fusion protein, creating a complex is formed, which has the fusion protein and lysozyme. For purification of the fusion protein, the complex can be separated from the remaining sample.

As described above, the corresponding fusion protein can be easily purified using lysozyme as the affinity ligand. The lysozyme used may be a c-type, g-type or i-type lysozyme. Preferably, the lysozyme is selected from the group of c-type lysozymes which have the ability to cleave beta- (1, 4) -glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine within peptidoglycans. Most preferably, the c-type lysozyme is derived from a mammal, bird or reptile, more preferably from a bird and most preferably from chickens. The lysozyme used as affinity ligand can be obtained from natural sources or can also be prepared recombinantly.

In one embodiment, lysozyme is added to a sample comprising the fusion protein. The sample may be, for example, a lysate, preferably a clarified lysate, or a culture medium comprising the fusion protein. The addition of lysozyme to the sample results in the fusion protein being precipitated in the form of a complex comprising the fusion protein and lysozyme. Accordingly, lysozyme is used in an amount or concentration so that precipitation of the complexes occurs. This probably leads to a conformational change, which favors the precipitation. This embodiment, in which the lysozyme is added to the sample in free form, has the advantage that the fusion protein can be purified without the use of a solid matrix often used in conventional affinity chromatography based on the use of affinity ligands , Therefore, this embodiment of the purification method of the present invention, which does not use a solid support for purification, allows the cost-conscious purification of a protein of interest by utilizing the fact that lysozyme can precipitate a fusion protein, provided that the fusion protein has an affinity tag, in particular a CDR3 region of one Heavy-chain antibody that binds lysozyme.

In one embodiment, the lysozyme used as the affinity ligand is immobilized to a solid support. Again, a complex is formed on the carrier. A corresponding purification method can be carried out according to the well-known principle of affinity systems (such as His-tag based systems) in which the target is bound by means of affinity ligands immobilized on columns. Lysozyme can be bound directly or, for example, via a linker molecule to the solid support. Suitable solid supports that can be used in a corresponding affinity chromatography purification process, such as columns or particles, are known in the art and therefore need no further description. The remaining sample can then be separated from the complex formed. If desired, the complex may optionally be washed prior to separation of the fusion protein from the affinity ligand lysozyme. According to a preferred embodiment, the fusion protein is separated from the lysozyme and thus released from the complex. In one embodiment, the release, also referred to herein as "elution," is achieved through the use of an elution solution, which can be achieved in a variety of ways, for example, sugars, peptides, free-lysozyme-binding affinity tags, and other agents can be used which have an affinity for the binding site of lysozyme and therefore can displace the fusion protein bound via the affinity tag from the complex According to an embodiment which allows a very mild elution of the fusion protein, an elution solution containing at least one sugar is used This embodiment is particularly useful when using an affinity tag that binds to the active site of lysozyme Preferably, such an affinity tag is a CDR3 region that is derived from a lysozyme As described above heavy chain antibody is derived Referenced the above disclosure, which also applies here. The elution solution may contain the natural substrate of the enzyme, for example selected from the group of peptidoglycans, in particular from the group of peptidoglycans having 1,4-beta linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues. Preferably, the sugar is in excess in the eluting solution to promote the release of the affinity tag from the active site of the lysozyme. This is particularly preferred when using an affinity tag that binds to the active site of lysozyme, as the excess of sugar can easily displace the fusion protein from the lysozyme, thereby releasing the fusion protein from the complex.

Further provided by the present invention is a complex comprising lysozyme and a fusion protein having a protein of interest and a lysozyme binding affinity tag. An appropriate complex can, for example, in an advantageous manner to

Crystallization of the contained fusion protein can be used to allow the analysis of the protein of interest. Lysozyme is a protein that can be easily crystallized, and has been found to be useful in promoting the crystallization of a fusion protein that has a protein of interest and a lysozyme-binding affinity tag. These features even allow the crystallization of a

Proteins of interest that are otherwise difficult or impossible to crystallize. The complex according to the present invention can therefore also be used for this purpose. The above-described use of a lysozyme-binding CDR3 region of a heavy chain antibody as an affinity tag is a preferred embodiment which has the advantage that the affinity tag is very small, thereby lowering the risk is that the three-dimensional structure of the protein of interest is changed. Suitable lysozyme binding sequences have been described above.

Another aspect of the present invention relates to the use of lysozyme to purify a fusion protein having a protein of interest and a lysozyme binding affinity tag. The details of the corresponding cleaning method are described above and reference is made to the above disclosure. Further, the present invention relates to the use of lysozyme for the precipitation of a fusion protein having a protein of interest and an affinity tag that binds lysozyme. As shown in the Examples, binding of the lysozyme-binding affinity tagged fusion protein to lysozyme results in conformational changes that result in reduced solubility of the lysozyme-fusion protein complex such that the complex then precipitates. The precipitation is fast and comprehensive. Therefore, the reduced solubility of the protein complex can be easily utilized to remove the complex from the remainder of the sample by various means, such as. As centrifugation, sedimentation u.ä. separate. As described above, appropriate precipitation-based purification is very cost effective and time-saving, and allows purification of the fusion protein without the need for affinity-based column chromatography.

Furthermore, an appropriate precipitation method for the detection of a protein, for example, in an assay of advantage. Here, an appropriate precipitation can be used, for example, to increase the local concentration of the protein to be detected and thereby the sensitivity of the assay. It can also be used to quantify a protein of interest by adding a second enzymatic or biochemical reaction to the precipitation process described above. Corresponding methods can also be used for diagnostic purposes.

Examples of the use of the present invention in diagnostic assays include, for example, the determination of the concentration of a protein or compound of interest in clinical samples, such as enzymes indicative of certain types of disease, inflammatory or marker proteins from pathogens, such as z. As viruses, which are usually present in low concentrations. One

Precipitation process according to the invention would therefore increase the sensitivity of assays of this particular type.

The fusion protein preferably has one or more of the features explained above. Reference is made to the corresponding disclosure. The protein of interest can be of any kind. The term "protein" refers to a molecule comprising a polymer of amino acids linked together by peptide bonds The term "protein" encompasses polypeptides of any length (e.g., greater than 50 amino acids) and peptides (eg, 2-49 amino acids). The term includes polypeptides and / or peptides having any activity or bioactivity including, for example, bioactive polypeptides, such as e.g. , Enzymatic proteins or peptides (e.g., proteases, kinases, phosphatases), receptor proteins or peptides, transporter proteins or peptides, bactericidal and / or endotoxin binding proteins, structural proteins or peptides, immune polypeptides, toxins, antibiotics, hormones, growth factors, vaccines and the same. The polypeptide may be selected from the group consisting of peptide hormones, interleukins, tissue plasminogen activators, cytokines, immunoglobulins, in particular antibodies or antibody fragments or variants thereof. The immunoglobulin can be of any isotype. Very often, IgG molecules (eg IgG1) are produced or needed as therapeutic proteins. An antibody fragment is any fragment of an antibody that has at least 20 amino acids of total antibody, preferably at least 100 amino acids, and that has the ability to bind an antigen. The antibody fragment may, for example, include the binding region of an antibody, such as. B. a Fab fragment, an F (ab) 2 fragment, multiple bodies comprising multiple binding domains, such as. As diabodies, triabodies or tetrabodies, single domain antibodies or Affibodies. An antibody variant is, for example, a derivative of an antibody or antibody fragment having the same binding function but, for example, an altered amino acid sequence. The antibody and / or antibody fragment may comprise a murine light chain, a human light chain, a humanized light chain, a human heavy chain and / or a murine heavy chain, as well as active fragments or derivatives thereof. Thus, it may be, for example, murine, human, chimeric or humanized. Preferably, the protein of interest is not initially associated with the affinity tag used in the fusion protein and is preferably not a heavy chain antibody or fragment thereof when a CDR3 region derived from a heavy chain antibody is used as the affinity tag ,

EXAMPLES

example 1

The affinity of the lysozyme-binding peptides provided by the present invention which can be used as an affinity tag was examined. The amino acid sequence of the affinity tags P2-P6 corresponds to the sequences shown as SEQ ID NO: 2-6 and is derived from a CDR3 region of a heavy chain antibody. Affinity tags P2 through P6 were N-terminally fused to GFP as a model protein of interest to produce a fusion protein having GFP (protein of interest) and a lysozyme binding affinity tag (P2 to P6). The GFP protein additionally had a His tag at the C-terminus. The affinity of the obtained GFP fusion proteins for lysozyme was determined. The results of the measurements are shown in the table below:

The results show that the small affinity tags according to the present invention consisting of or derived from the CDR3 region of a heavy chain antibody, despite being stabilized not by frameworks or other stabilizing structures, Lysozyme can bind with high affinity. The affinity can be further increased by appropriate amino acid substitutions in the peptide sequence of the affinity tag. This can be supported, for example, by molecular modeling.

Example 2:

Example 2 shows that the lysozyme-binding affinity tag according to the present invention

Invention can be used for precipitation and thus for the purification of a fusion protein having a protein of interest and the affinity tag. The fusion protein P5-GFP has a lysozyme-specific affinity tag at the N-terminus. In the affinity tag P5 (see SEQ ID NO: 5), a cysteine was replaced with a serine as compared with the affinity tag P4 (see SEQ ID NO: 4). The fusion protein

P5-GFP additionally had a C-terminal His tag and was expressed in E. coli. Subsequently, lysozyme was used to precipitate the P5-GFP fusion protein. Two samples were tested. One sample had the clarified bacterial lysate (Sample 1), while the other sample contained P5-GFP pre-cleaned and concentrated using Ni-NTA Superflow Resin (Qiagen) according to the manufacturer's instructions (Sample 2). Subsequently, the mixture was centrifuged. As shown in Figure 1, lysozyme precipitates the P5-GFP fusion protein. The binding of lysozyme to the affinity tag of the P5-GFP fusion protein results in a complex that precipitates due to a conformational change of the lysozyme. The precipitated complex is visible in FIG. 1 as a yellow pellet. Sample 2, which contained the P5-GFP fusion protein pre-purified via the additional His tag, resulted in a larger pellet due to the higher concentration of the fusion protein. As shown in Figure 1, the addition of lysozyme to the clarified lysate also precipitates the fusion protein, thereby allowing for rapid and easy isolation of the fusion protein.

The resulting P5-GFP-lysozyme complexes were then purified by gel filtration and analyzed by SDS-Page and Coomassie staining and photometric analysis of individual fractions at OD = 509 nm. Figure 2 shows the photometric analysis of individual fractions purified by gel filtration. Relative fluorescence was plotted as a function of wavelength. The highest absorbance was observed in agreement with the excitation emission spectrum of GFP at OH = 509 nm. Various concentrations were tested. The results show that at a certain concentration the immediate precipitation of the complexes occurs. This precipitates over 90% and even over 95% of the protein of interest.

Example 3:

Here, the use and specificity of the lysozyme affinity tag (lyso tag) for protein purification was determined. 1000 μΙ GFP-His, GFP-Strep and P5-GFP buffered in 100 mM NaCl, 50 mM Tris, pH = 7.5 were added to Eppendorf tubes, then lysozyme was added and the samples were incubated at room temperature. The addition of lysozyme resulted in increased turbidity of the P5-GFP sample, which was not observed in the GFP-His and GFP-Strep samples. Subsequently, the samples were centrifuged for 15 minutes at 4 ^< Ό, resulting in pelleting of the sample containing P5-GFP, but not for GFP-His and GFP-Strep. The results are shown in Fig. 3, the specificity of the precipitation of P5-GFP by

Addition of lysozyme occupied. (1) GFP-His, (2) GFP-Strep, (3) P5-GFP. A: Samples before adding lysozyme. B: Samples after the addition of lysozyme. The P5-GFP sample appears turbid after the addition of lysozyme. C: Samples after centrifugation at 12,000 g at 4 ^< Ό. The addition of lysozyme resulted in the precipitation of the P5-GFP sample as seen on the yellowish pellet after centrifugation. The pellet contains the formed

Complex. No effect was observed in the control samples. The His-GFP sample was even after centrifugation, the Strep-GFP sample showed a whitish pellet after centrifugation. This demonstrates that precipitation was specifically caused by the interaction of the lysozyme with the P5 tag and was not induced by nonspecific protein aggregation, for example, caused by the addition of lysozyme to the sample. It should be noted that the GFP is not denatured during the precipitation process and retains its fluorescence throughout the purification process. In the supernatant, GFP was also barely detectable, which underlines the efficiency of the method. Thus, a fast, inexpensive and effective cleaning process is provided, which is feasible without support materials such as columns.

Example 4:

Further investigations were carried out to show that the precipitation is not caused by a non-specific interaction between lysozyme and GFP, but is due to a specific interaction between the lysozyme and the affinity tag. Further, it should be shown that the concept of the present invention can be implemented with various proteins of interest.

In Example 4, interferon-alpha was used as the protein of interest N-terminally provided with the lysozyme-binding affinity tag P5 (see SEQ ID NO: 5). This yielded a fusion protein comprising interferon alpha (protein of interest) and a lysozyme binding affinity tag (P5). Addition of lysozyme resulted in immediate precipitation of the lysozyme-binding affinity tag-bearing fusion protein. The precipitation of the complex was assisted by centrifugation to produce a type of pellet containing the complexes formed. The results of

Gel electrophoresis are shown in FIG.

In the first lane (next to the marker), the sample containing lysozyme and the tagged fusion protein (before centrifugation) was loaded onto the gel. As can be seen, the sample comprises lysozyme and the affinity tagged one

Fusion protein. In the third lane next to the marker (the second lane is free), the supernatant was applied. As can be seen, the supernatant comprises excess lysozyme and only small residual amounts of the fusion protein. This shows that the addition of lysozyme effectively precipitated nearly all of the fusion protein containing interferon-alpha. Next, various concentrations of the pellet were applied to the gel. As can be seen, the precipitated pellet comprises the interferon-alpha-containing fusion protein and lysozyme.

Claims

1 . A fusion protein comprising a protein of interest and an affinity tag that binds lysozyme.

2. A fusion protein according to claim 1, wherein the affinity tag of the fusion protein has one or more of the defined in claims 8 to 1 1 features of the CDR3 region.

A method for purifying a fusion protein according to claim 1 or 2 from a sample comprising the fusion protein, wherein the method employs lysozyme as the affinity ligand, and wherein the affinity tag of the fusion protein binds to lysozyme to form a complex.

The method of claim 3, wherein a) the lysozyme is contacted with the sample so as to precipitate the complex comprising the fusion protein and lysozyme, or

b) the lysozyme is immobilized on a carrier and the fusion protein is bound to the carrier via the lysozyme.

5. The method according to claim 3 or 4, wherein the remaining sample is separated and preferably the fusion protein is released from the complex.

6. The method according to one or more of claims 3 to 5, wherein the

Fusion protein is released using an elution solution having a sugar that is bound by lysozyme.

7. A binding agent that specifically binds to an objective compound, wherein the binding agent has a CDR3 region derived from a heavy chain antibody but has no framework regions or other elements to stabilize the CDR3 region and wherein the binding agent crosses the target compound binds to the CDR3 region.

A binding agent according to claim 7, wherein the binding agent consists of the CDR3 region.

9. binding agent according to claim 7 or 8 with one or more of the following features: a) the CDR3 region of the binding agent binds the target compound with the same specificity and / or affinity as a heavy chain antibody having the framework regions;

b) the CDR3 region has a length of 10 to 30, preferably 12 to 25, amino acids;

c) the CDR3 region binds to a groove, pocket or canyon of a target compound;

d) the CDR3 region contains no amino- and / or carboxy-terminal amino acid residues that would allow cyclization;

e) the CDR3 region mimics the natural substrate binding to the pocket, groove or canyon of the target compound to which the CDR3 region binds, and / or

f) the CDR3 region has no cysteine residue.

10. binding agent according to one or more of claims 7 to 9, wherein the binding agent via the CDR3 region lysozyme binds as a target compound.

1 1. The binding agent according to claim 10, wherein the binding agent has a CDR3 region which has a sequence selected from the group consisting of SEQ ID NO: 1 to 6 or consists of one of these sequences.

12. A fusion protein comprising a protein of interest and an affinity tag, wherein the affinity tag has a CDR3 region derived from a heavy chain antibody but has no framework regions for stabilizing the CDR3 region, wherein the CDR3 region preferably one or more of the in the

Claims 8 to 1 1 has defined features of the CDR3 area.

13. Use of lysozyme for the purification, precipitation or support of the crystallization of a fusion protein according to claim 1 or 2.