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EP3201328A1 - Protéine de fusion et procédé de purification - Google Patents

Protéine de fusion et procédé de purification

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Publication number
EP3201328A1
EP3201328A1 EP15771867.7A EP15771867A EP3201328A1 EP 3201328 A1 EP3201328 A1 EP 3201328A1 EP 15771867 A EP15771867 A EP 15771867A EP 3201328 A1 EP3201328 A1 EP 3201328A1
Authority
EP
European Patent Office
Prior art keywords
lysozyme
fusion protein
cdr3 region
protein
binding agent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP15771867.7A
Other languages
German (de)
English (en)
Inventor
Johann Kubicek
Frank Schäfer
Jörg LABAHN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP3201328A1 publication Critical patent/EP3201328A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2462—Lysozyme (3.2.1.17)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01017—Lysozyme (3.2.1.17)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an object of the present invention is to provide means for assisting the crystallization of a protein of interest.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a host cell is provided which has a corresponding expression vector.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the CDR3 region may form convexities or protrusions that may occupy, for example, the groove of a target compound.
  • 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.
  • 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.
  • the CDR3 region is not limited by intramolecular bonds, e.g. Disulfide bonds, cyclized.
  • 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.
  • the CDR3 region does not contain cysteine residues.
  • 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.
  • 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.
  • an amino acid is selected for substitution, which maintains or even improves the binding ability.
  • the binding agent is the CDR3 region.
  • 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.
  • 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.
  • 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.
  • the binding agent binds lysozyme as the target compound across the CDR3 region.
  • the binding agent may also consist of the CDR3 region.
  • the binding agent comprises a CDR3 region having or consisting of a sequence selected from the group consisting of
  • 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.
  • 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.
  • 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.
  • a recombinant fusion can be achieved, for example, by expressing the protein of interest and the affinity tag as a fusion construct.
  • 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.
  • the CDR3 region also has no other stabilizing structures.
  • the CDR3 regions derived from corresponding heavy chain antibodies have several advantages that make them particularly suitable for use as an affinity tag.
  • 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.
  • 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.
  • 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.
  • a fusion protein comprising a protein of interest and an affinity tag capable of binding lysozyme.
  • 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.
  • the lysozyme-binding affinity tag is located at either the N-terminus or the C-terminus of the fusion protein.
  • the affinity tag is according to the present invention
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the affinity tag comprises or consists of an immunoglobulin molecule or a functional fragment thereof that binds lysozyme.
  • the immunoglobulin molecule may be an antibody.
  • antibody refers to a protein having at least two heavy chains and two light chains linked by disulfide bonds
  • 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.
  • CDRs complementarity determining regions
  • FR framework regions
  • 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.
  • 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.
  • a fragment or derivative of an antibody as used herein generally refers to a functional fragment or derivative capable of binding to the antigen.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the affinity tag has a sequence selected from the group consisting of
  • Lysozyme and are thus suitable for use as an affinity tag.
  • SEQ ID NO: 5 a cysteine was changed to a serum as compared with SEQ ID NO: 4.
  • the fusion protein according to the present invention has a proteolytic cleavage site.
  • the proteolytic interface separates the
  • 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.
  • 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.
  • the present invention relates to an expression vector for expressing a fusion protein having a protein of interest and a lysozyme-binding affinity tag.
  • 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.
  • Signal sequence is usually cleaved, so that then the fusion protein is secreted.
  • 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.
  • 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.
  • 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.
  • a wide variety of transcriptional and translational regulatory sequences may be employed.
  • 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.
  • other regulatory signals may also be used.
  • the design of corresponding expression vectors is known to the person skilled in the art.
  • 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.
  • 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.
  • eukaryotic host cells can be used, such.
  • 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.
  • 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.
  • 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.
  • a host cell which has an expression vector according to the invention. This has been described in detail above and reference is made to the corresponding disclosure.
  • 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.
  • 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
  • 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
  • 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.
  • the complex can be separated from the remaining sample.
  • 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.
  • the lysozyme used as the affinity ligand is immobilized to a solid support.
  • 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.
  • the complex may optionally be washed prior to separation of the fusion protein from the affinity ligand lysozyme.
  • the fusion protein is separated from the lysozyme and thus released from the complex.
  • 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
  • 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
  • such an affinity tag is a CDR3 region that is derived from a lysozyme As described
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an appropriate precipitation method for the detection of a protein for example, in an assay of advantage.
  • 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.
  • 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.
  • 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 e.g., receptor proteins or peptides
  • transporter proteins or peptides e.g., 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.
  • it may be, for example, murine, human, chimeric or humanized.
  • 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 ,
  • 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:
  • Example 2 shows that the lysozyme-binding affinity tag according to the present invention
  • the 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.
  • 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).
  • 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.
  • lysozyme affinity tag lyso tag
  • 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.
  • 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
  • the sample containing lysozyme and the tagged fusion protein was loaded onto the gel.
  • the sample comprises lysozyme and the affinity tagged one
  • the supernatant was applied in the third lane next to the marker (the second lane is free). 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.

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Abstract

L'invention concerne entre autres une protéine de fusion comprenant une protéine d'intérêt et une étiquette d'affinité qui se lie au lysozyme. Le lysozyme peut être employé pour purifier, précipiter ou favoriser la cristallisation d'une telle protéine de fusion. De plus, l'invention concerne un agent de liaison qui se lie spécifiquement à un composé cible, l'agent de liaison présentant un domaine CDR3 qui est dérivé d'un anticorps à domaine individuel mais ne présente pas de domaine de squelette ou d'autres éléments pour la stabilisation du domaine CDR3, et l'agent de liaison se liant au composé cible par le biais du domaine CDR3.
EP15771867.7A 2014-09-05 2015-09-07 Protéine de fusion et procédé de purification Withdrawn EP3201328A1 (fr)

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DE102014217793.6A DE102014217793A1 (de) 2014-09-05 2014-09-05 Fusionsprotein und Aufreinigungsverfahren
PCT/EP2015/070393 WO2016034741A1 (fr) 2014-09-05 2015-09-07 Protéine de fusion et procédé de purification

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