Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
• • 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
  • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
  • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
  • C07K1/1075—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of amino acids or peptide residues
• • 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
  • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
  • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
  • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• • 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
  • C07K14/745—Blood coagulation or fibrinolysis factors
  • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• • 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
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
  • C12N2710/22022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• the present invention relates to a method for connecting two or more molecular substances via adapter segments, which bring about a directed interaction, based on the affinity of proline-rich amino acid sequences and protein domains of the WW type.
• the interaction of two or more molecular substances is a common problem in the context of biotechnological and pharmaceutical-medical research, development and application.
• the interactions of two or more proteins or peptides are usually considered as molecular substances.
• Such interactions are often investigated in the context of biochemical and cell biological research, for example for intra- and intercellular communication, signal transduction at the molecular level or when analyzing protein-protein interactions (including when using the two-hybrid systems and methods derived from them) .
• the association of biomolecules, in particular two or more proteins, for in-vitro synthesis of a fusion protein is of great importance for many biotechnological processes.
• Fusion proteins generated in this way can be, for example, heterobifunctional (bivalent) antibodies (so-called diabodys; see O. Perisic, PA Webb, P. Holli ger, G. Winter & RL Williams, Crystal structure of a diabody, a bivalent antibody fragment, Structure 2, pp. 1217-1226, 1994), which consist of the binding domains (Fab / Fv / scFv fragments) of two different antibodies. If the two valences are each directed against tumor cells or natural killer cells, for example, the bivalent, hybrid fusion protein can accordingly mediate the attachment of killer cells to tumor cells.
• bivalent, hybrid fusion protein can accordingly mediate the attachment of killer cells to tumor cells.
• Heterobifunctional constructs are often generated by the synthesis of a fusion protein at the gene level. This usually requires suitable linkers between the two partners, as well as accessible terms of the polypeptide chains. In unfavorable cases, the fusion of the partners can result in the fusion product being inactive, for example because the fusion protein cannot form a correct three-dimensional folding topology. It is therefore often desirable that the two be put together Fusion partners in vitro, that is, after the separate synthesis and folding of the two partners takes place. Such a method would also allow, for example, the rapid production and analysis of various combinations of individual building blocks without the need for new genetic constructs.
• adapter segments are required, through which the process of the fusion or the directed association of the partners involved is decoupled from their production. Furthermore, it is necessary for the adapter segments (domains or peptide sequences) to be firmly coupled to the partners involved without otherwise changing their specific properties.
• Peptides and small protein domains play a particularly important role here, since they can be attached to the desired target proteins comparatively easily in the process of recombinant protein production.
• Applications for this are, for example, the purification of recombinantly produced proteins via specific binding segments.
• a poly-histidine peptide section is often used here for binding to nickel chelate columns (cf. P. Hengen, Purification of His-Tag fusion proteins from Escherichia coli, Trends Biochem. Sci. 20, pp. 285-286, 1995) , or the binding of a peptide segment known as Strep-Tag to streptavidin (TG Schmidt, J.
• the His-Tag method has the disadvantage, however, that only the poly-histidine peptide section can be bound to structures containing nickel ions; the connection of two natural proteins or peptides, for example, is not possible in this way. The method is therefore not suitable or only suitable in exceptional cases for the connection of molecular substances. In the preparations cleaned in this way, nickel ions are often found in the solution, which makes the system unattractive for medical-therapeutic use.
• the region of the binding partner which mediates the binding is relatively large, so that it is not suitable for many compounds for steric reasons.
• avidin and streptavidin each have four binding sites, so that it is very difficult to form two different interconnected molecular substances in solution in a controlled manner.
• the immobilization of the proteins on a solid, inert matrix is of great biotechnological importance, for example in the refolding of proteins on a matrix to prevent aggregation processes during folding (cf. G. Stempfer, B. Höll-Neugebauer & R. Rudolph, Improved refolding of an immobilized fusion protein, N ⁇ t.
• the object of the present invention is to provide a method for connecting molecular substances which does not have the disadvantages of the prior art mentioned.
• a method according to claim 1 for connecting two or more molecular substances to one another via adapter segments characterized in that one of the molecular substances is modified in such a way that it as an adapter segment derives a WW domain or at least one region thereof Structure, another molecular substance is modified so that it has as an adapter segment in at least one region a proline-rich sequence that binds to the WW domain or a structure derived therefrom, and the molecular substances by the assembly of the WW domain or thereof derived structures and proline-rich sequence interact with each other to achieve a bond to each other.
• the linking of two or more different molecular substances (molecular species) to a - usually heterobifunctional - fusion construct is a process of high biotechnological and pharmaceutical interest.
• proteins and / or peptides are usually used as the molecular species to be connected, since the adapter segments of the present invention originate from this chemical class.
• other molecular substances which are provided with one of the adapter segments of this invention can also be used according to the invention.
• a solid matrix can be connected to a molecular substance via the adapter segments mentioned. Often the two substances to be connected have to be connected stably and covalently to one another.
• a molecular species has to be immobilized for a limited time, that is to say interact specifically with a matrix, for example to purify a protein from a crude cell extract in the recombinant production of a protein, or in a matrix-assisted refolding of the protein.
• the present invention is suitable for such applications.
• a protein can be directed into a virus-like envelope for packaging, or two or more different proteins can be combined to form a chimeric protein with new properties, for example as bivalent antibodies.
• this interaction can be used to immobilize a molecular species, for example to separate this substance from a mixture of substances.
• the molecular substances can be covalently bound to one another.
• This covalent bond can be bridged by disulfide bridging by artificially introduced cysteines at a suitable location in both molecular substances to form a permanent connection between the two molecular lead laren substances.
• the disulfide bridge can be used to generate bifunctional fusion molecules which are stable under physiological and all customary solvent conditions and can therefore also be used for medical, therapeutic, diagnostic and biotechnological processes.
• the highly specific interaction of protein segments which are known under the term WW domains, with a proline-rich peptide sequence (with a proline content of more than 50% within a short peptide sequence of 2 to 6) is used to connect two or more molecular substances to one another Amino acids).
• These two species of molecules show an extremely strong interaction (K D 20 to 100 nM) when they are incubated with each other.
• the slow dissociation of the partners means that the interaction is initially only temporary. If this is undesirable, the dissociation can be prevented by fixing the binding partner with a disulfide bridge.
• cysteines are artificially introduced into the two adapter segments or in the region of the adapter segments in a suitable spatial position. After the partners have been associated, the cysteine pairs can be oxidized by selecting suitable redox conditions and thus permanently covalently linked to one another.
• the resulting hybrid fusion protein can have essential properties of the underlying molecular species.
• the WW domain is a small, globular protein domain that usually consists of 30 to 40 amino acids (see M. Sudol, The WW Domain Binds Polyprolines and is Involved in Human Diseases, Exp. & Mol. Medicine 28, pp. 65- 69, 1996), but shorter variants are also known.
• WW domains have a high natural affinity for proline-rich ligands that are bound with dissociation constants from 20 to 100 nM.
• the proline-rich ligands have a minimum length of 5 to 15 amino acids necessary for binding with a proline content of more than 50% within this segment, with the direct interaction usually within a local segment of 2 to 6 amino acids (with more than 50% proline content ) occurs. Natural ligands are almost there only proteins that contain proline-rich sections in their amino acid sequence, but proline-rich peptides are also specific ligands of WW domains.
• WW domain is derived from the observation that two conserved tryptophan residues (WW for short) occur at intervals of 20 to 22 amino acids; the second tryptophan and a series of mostly also conserved hydrophobic amino acids form the binding pocket for the proline-rich ligand. After the second tryptophan, a conserved proline is often located at a distance of 2 amino acids.
• WW tryptophan residues
• a number of different WW domain types are known, which are currently classified into 4 classes and which differ from one another in particular with regard to the preferentially bound proline-rich peptide ligands.
• WW domains can in principle compete with the (structurally unrelated) SH3 domains for the binding of proline-rich ligands, but the ligands of the SH3 domains have different consensus sequences, so that proline-rich peptide ligands can be derived which are specifically bound by WW domains , In addition, the binding of the WW domains to proline-rich ligands is generally stronger than that of SH3 domains.
• the table below provides an overview of representatives and ligand binding properties of WW domain proteins.
• Type 1 Pro-Pro (any) - (Tyr) YAP65, Pinl, Dystrophin
• the conversion of a WW domain of type I into a type II, combined with the associated change in the specificity with regard to the proline-rich peptide, can be achieved, for example, by the amino acid exchanges L14W and H16G in the WW type I domain sequence ,
• the structure of a representative from class I shows that this WW domain consists of three ß-strands that form a ß-sheet (see M Macias, M Hyvonen, E Baraldi, J Schultz, M Sudol, M Saraste & H Oschkinat, The Structure of the WW Domain in Complex with a Proline-Rich Peptide, Nature 382, S 646-649, 1996)
• the ligand binding pocket is formed from the second ⁇ -strand of the leaflet with the participation of the second conserved tryptophan
• WW domains are also directly or indirectly associated with a number of diseases, such as congenital Liddle syndrome, muscular dystrophy, and Alzheimer's disease hence the goal of a number of therapeutic strategies. Finally, WW domains play a biological role in the embryonic development of the kidneys and in the intracellular life cycle of retroviruses
• WW domains can surprisingly form a stable structure (folding topology) under customary solvent conditions, even if they are isolated from their original molecular context and genetically fused in or to other proteins, such as a viral coat protein
• Linker segments consisting of the amino acids serine and glycine, apparently neither fold their proteins in external loops of proteins, nor is the binding property of the WW domain negatively influenced thereby.
• variants of the WW domain ne applies in which, for example, amino acids have been exchanged for cysteine at specific positions. It can also apply to other structures derived from WW domains, such as, for example, several WW domains which are consecutively lined up, the contributions to the binding of which add up or are favorable in favorable cases, shortened or extended WW domains, or also WW domains with location-specific exchange of individual amino acids, which - depending on the desired application dung - for example stronger or weaker than the natural protein domains are able to bind to proline-rich sequences. Such changed domains can be obtained, for example, by binding screening using the phage display technology according to the prior art.
• Proteins that have an inserted that is, inserted into suitable loop areas within the polypeptide chain of the host protein) or fused (each located at the N-terminus and or at the C-terminus of the host protein) show a high affinity for proline-rich sequences.
• These proline-rich sequences can in turn be fused to other proteins, peptides or other molecular substances. Any two molecular species can thus be attached to one another by the interaction partners attached to them (WW domain and proline-rich sequence). This attachment is based initially on a hydrophobic interaction, mediated by WW domain and proline-rich ligand.
• this interaction can be adapted with regard to higher specificity and more flexible use by adding, for example, ionic interactions or introducing covalent bonds between the WW domain and the proline-rich ligand.
• the addition of the proline-rich ligand to the WW domain can be increased or made more specific by placing additional amino acids that are charged differently or by point mutations in or near the adapter segments.
• Covalent disulfide bridging of the two components in turn allows the adapter segments and the associated molecular substances to be bonded permanently and firmly.
• a connection of more than two molecular substances to one another is also possible according to the invention.
• the kinetic parameters of the interaction such as the dissociation constant (k D )
• k D dissociation constant
• the interactions between WW domains and proline-rich peptide sequence are fundamentally suitable for the use cases described in the present invention. Due to the nature of the interaction of the adapter segments, only a heteromeric hybrid species is formed, consisting of a part with a WW domain and a part with a proline-rich sequence. The formation of homofunctional molecules (homodimers) can be excluded. Compared to other systems with comparable properties, the WW domain used has the advantage of being exceptionally small and compact. As a result, it is clearly superior to other ligand-binding domains (for example lipocalines and anticalins) in many applications, for example the antigen-antibody interactions.
• ligand-binding domains for example lipocalines and anticalins
• cysteine residues at specific sites within the WW domain and in the proline-rich substrate can be used to effect the covalent coupling of the association partners, and consequently, beyond the interaction between the WW domain and the proline-rich sequence to bring the protein portions fused onto these adapter segments into a stable connection with one another.
• a dissociation of the interaction partners cannot take place under unfavorable conditions such as particularly high or very low salt concentrations or under physiologically extreme temperatures.
• an exchange for cysteine is carried out at point Asp8 (numbering follows the WW domain from the form-binding protein FBP11) or alternatively at point Lysl9. These positions are only selected as examples; the introduction of specific cysteines can also be useful and successful at other sites in the WW domain or in the vicinity thereof, or in the proline-rich sequence or in the vicinity thereof.
• heterobifunctional species are formed because the strong interaction of the proline-rich peptide and WW domain means that initially only associations between these two adapter segments can form.
• the subsequent disulfide bridging under oxidizing conditions then leads to the directed formation of covalently bridged, heteromeric species. Due to the high local concentration (approximation) of the cysteines in the associated form, the disulfide bridging can also be successful under slightly reducing conditions and can therefore be particularly specific.
• the method described in the present invention is suitable for attaching any interaction partners in solution (in vitro) to one another, both temporary and permanent binding of the two partners being possible.
• the method can also be used to specifically separate proteins or peptides or other molecular substances that are equipped with one of the two adapter types (WW domain or proline-rich sequence) from a mixture of substances. This is done by reversible binding to a matrix that has covalently bound the other interaction partner. The strong bond causes the molecules to adhere to the matrix even under stringent solvent conditions.
• the method thus allows, for example, the rapid and efficient purification of recombinant proteins from the cell extract of bacteria or eukaryotic cells, provided that the recombinant (to be purified) molecule is one of the two adapter segments (WW domain or proline-rich sequence) in fusion or as Insertion carries while the corresponding counterpart to the adapter segment has been immobilized on the solid phase.
• this immobilization method is suitable for carrying out specific modifications or refolding of the immobilized protein on the matrix while avoiding aggregation processes.
• the present invention also enables applications in which a simple and stable immobilization of a molecular substance plays a key role, for example in biosensors or in bioreactors (cf. RS Phadke, Biosensors and enzyme immobilized electrodes, Biosystems 27, pp. 203-206 , 1992; M. Abdul-Mazid, Biocatalysis and immobilized enzyme / cell bioreactors. Promising techniques in bioreactor technology, Biotechnology (NY) 1 1, pp. 690-695, 1993).
• proteins and peptides can also be used for the method described in the present invention.
• peptide derivatives peptide antibiotics, proteins with modified side chains such as fluorescent labeling, alkylations, acetylation, mixed disulfides with thiol-containing substances, and analogous changes can be used in the same way.
• peptide or protein conjugates with carbohydrate, nucleic acid or lipid components can also be used in the process.
• Nucleic acids such as DNA, RNA, ribozymes, synthetic nucleic acids such as peptide nucleic acids, or hybrids thereof can also be coupled to an adapter segment, for example by chemical means. They are then also suitable for interacting with an analogue interaction partner. The only requirement is the stable connection of one of the two adapter segments used.
• proteins in particular antibodies, antibody-analogous substances, enzymes, structural proteins, and capsomeres of viruses or phages are suitable as proteins.
• the insertion or binding of the proline-rich sequence or the WW domain or a structure derived therefrom into a molecular substance can in principle take place at any point in the molecular substance, provided that this does not significantly influence the structure of the WW domain. If appropriate, it is advantageous to carry out the attachment or insertion using suitable linker segments, as described in Example 1 for the PyVPl-WW150 protein. In the case of insertion into proteins, it is expedient to look for regions of the structure of the protein in which there are no periodic secondary structural elements such as a helix or a ⁇ -sheet.
• the insertion of WW domains or proline-rich sequences in protein structures takes place most advantageously where “turn” regions or “random coil” regions are present according to the usual definition.
• the binding of the two adapter segments to one another can be viewed in terms of different physical interactions.
• a hydrophobic effect can dominate in stabilizing the interaction, as is demonstrated in Example 7 below.
• other forms of interaction can also contribute to the formation of a bond, such as, for example, ionic interactions, ion-dipole interactions, dipole-dipole interactions, hydrogen bonds, van der Waals forces, or dispersion forces.
• a covalent connection of the two molecular sub- punching can be effected.
• a chemically stable atomic bond is formed between two atoms of the interaction partners, preferably in the form of a disulfide bridge from two cysteine side chains involved.
• the matrix can be used to immobilize one of the adapter segments (WW domain or proline-rich sequence), for example via the N-terminus of the proline-rich sequence or the WW domain (coupling via N-hydroxysuccinimide ester of the matrix) or via a thiol group of one in the proline-rich sequence or the WW domain containing cysteine (coupling via iodoacetamide group of the matrix).
• suitable matrices are agarose and agarose derivatives, agarose beads, sepharose, dextrans, carbohydrates or similar polymer material according to the prior art.
• FIG. 1 shows a schematic representation of the invention.
• Adapter segments based on the interaction of proline-rich substances with WW domains and forms derived from them are used, (a) linkage of two molecular species A and B via adapter segments, (b) linkage of two molecular species A and B analogous to (a ), but with additional disulfide bridging to covalently link the partners, (c) matrix immobilization of a molecular substance via the adapter segments (one of the molecular ones represents the matrix or part of the matrix).
• the adapter segments can be attached to the molecules both at the ends (termini) and in the form of insertions.
• FIG. 2 shows in (a) a comparison by means of SDS-PAGE of the protein masses and the cleaning efficiencies of different variants of the polyomavirus protein VP1, the PyVPl-CallS-T249C variant (comparable to the wild type of the protein) and the PyVPl-WW150 variant in which a WW domain is inserted in a loop near amino acid position 150.
• Production and cleaning of the variants is described in detail in Example 1. ben.
• degradation products of the protein which usually occur have a smaller molecular mass
• CD circular dichroism spectra
• the inserted WW domain at position 150 shows a native fold, which increases the proportion of ⁇ -leaflets in the CD spectrum.
• FIG. 3 shows the binding of PyVPl-WW150 to a sensor chip with immobilized proline-rich peptide, according to Example 2.
• the three measurements, based on surface plasmon resonance, show that the solvent additives have only a minor influence on the affinity and specificity of the interaction, the in (b) and (c) the additives used each represent complex physiological substance mixtures, (a) binding of PyVPl-WW150 to the sensor surface under usual solvent conditions, (b) binding of PyVPl-WW150 to the sensor surface using Dulbecco's PBS as eluent, ( c) Binding of PyVPl-WW150 to the sensor surface when adding fetal calf serum (FCS) as a model for a mixture of biologically relevant substances.
• FCS fetal calf serum
• FIG. 4 shows an SDS gel to show the specific binding of PyVPl-WW150 to a matrix containing proline peptides.
• Lane 1 order VP1-WW150 (cleaned according to example 1); Lane 2 and Lane 3: different wash fractions; Lane 4 and Lane 5: elution fractions with 1% SDS in the elution buffer; Lane 6: 10 kDa molecular mass standard.
• the example shows that WW domain-containing proteins can be reversibly immobilized on a matrix.
• the observed double band of the PyVP 1 variant represents the native protein and a proteolytic degradation band of the protein, which usually occurs in all preparations.
• FIG. 5 shows a gel filtration (TSKGel G5000PWXL, from TosoHaas) to demonstrate the binding of a proline-rich peptide to the surface of a virus-like capsid, the WW domains inserted into the coat protein VP1 being located on this surface.
• the PyVPl-WW150 protein is assembled to the capsid under the conditions given in Example 4.
• a proline-rich peptide can be bound to the virus-like capsids and is detected by the specific absorption of a dye coupled to it.
• FIG. 5 a detection of capsid formation by absorption at 260 and 280 nm; the capsids elute at a volume of 6 to 8 ml, the unassembled, free pentamers appear at 9 to 10 ml.
• FIG. 5b absorption of the fluorescence-labeled peptide at 490 nm; the elution is carried out in parallel with the capsule elution and the pentamer elution at 6 to 10 ml. Above 10 ml, excess, free peptide and the fluorescent dye elute.
• FIG. 5c the free, unbound peptide shows no interaction with the matrix and elutes only above 10 ml.
• FIG. 5d superimposition of chromatograms 5a and 5b, to show the co-elution of the bound peptide with the capsid fraction ,
• Figure 6 shows in (a) the cleaning of the variants PyVPl-3C-WW1 and PyVPl-3C-WW [N-14].
• the SDS gel (12%) shows the PyVP 1 protein without WW domain (lane 2), the PyVPl-WW150 variant from example 1 (lane 3), and the two variants from example 8 (PyVPl-3C-WWl on lane 4 and lane 5, PyVpl-3C-WW [N-14] on lane 6).
• Lanes M molecular mass standard (10 kDa ladder
• Lane Int wash fraction from the Intein affinity column
• Fractions 1 to 9 different elution fractions of the GFP-PLP protein.
• FIG. 7 shows the packaging of molecular substances in the interior of virus-like casings based on polyomavirus VP1 variants, (a) packaging of GFP-PLP in casings which contain PyVPl-3C-WW1. GFP-PLP is added in 6-fold molar excess under standard conditions before assembly.
• a gel filtration sex experiment (TSKgel G6000PWXL, TosoHaas) is shown, in which capsid fractions (elution at 9 ml) are separated from free, unassembled pentamers of the PyVP 1 variants and the GFP protein (1 1 to 13 ml).
• a detectable amount of GFP is present in the capsid fraction, which was directed into the interior of the capsids via the WW domains / polyproline interaction, (b) packaging of GFP with WW domain at the N-terminus into the interior of virus-like capsids, assembled from PyVPl-3C- [N-14] -PLP (proline-rich sequence at the truncated N-terminus).
• GFP-WW1 is incubated with PyVPl-3C- [N-14] -PLP and capsids are produced by assembly under standard conditions.
• the polyprolm peptides are brought into the interior of the capsids via the affinity for the WW domain, (c) packaging of a fluorescence-labeled peptide (proline-rich sequence) into the interior of virus-like capsids.
• the peptide is incubated with PyVPl-3C-WW [N-14] and capsids are produced by assembly under standard conditions.
• the polyproline peptides are brought into the interior of the capsids via the affinity for the WW domain, (d) packaging GFP with a proline-rich sequence at the C-terminus into the interior of virus-like capsids which have been labeled with PyVPl-3C-WW [N -14] are assembled.
• GFP-PLP is incubated with PyVPl-3C-WW [N-14] and capsids are produced by assembly under standard conditions.
• the GFP-PLP is brought into the interior of the capsids via the affinity for the WW domain.
• FIG. 8 shows an SDS gel to illustrate the purification of proteins with a WW domain from a mixture of substances, here a cell extract.
• Lane 1 10 kDa molecular mass standard
• Lane 2 crude extract of (PyVP 1-3 C-WW1) -eintein-CBD fusion protein
• Lane 3 run
• Lane 4 to 10 Different fractions of the elution of the fusion protein, with 2% SDS in the elution buffer.
• the fusion protein is immobilized on a column with covalently bound proline-rich peptide.
• the column is washed with a total of 10 column volumes of a buffer containing 2 M NaCl.
• degradation products thereof and molecular chaperones which are known to bind to PyVPl, are detected.
• FIG. 9 shows an HPLC fluorimetric representation of the disulfide bridging of a proline-rich molecular substance with a WW domain, which was fused to gluthathione-S-transferase (GST) for the purpose of affinity purification (FIG. 9a) and under reducing conditions (50 mM DTT, Fig. 9b).
• GST gluthathione-S-transferase
• a linker consisting of alternating Gly-Ser amino acids is additionally inserted before and after the WW domain.
• the viral coat protein used in the given example is the polyomavirus VP1 coat protein pentamer in solution, which according to the prior art can be assembled in vitro into a virus-like coat. From the crystal structure of the protein it can be seen that a loop region in the structure in the vicinity of amino acid position 150 may be suitable for the insertion of the WW domain, since this loop region turns into a virus-like envelope when the pentamer protein is assembled the outside of the case.
• PyVPl-WW150 takes place as a fusion protein with a C-terminally fused protein domain and an adjoining chitin binding domain (CBD).
• CBD chitin binding domain
• a plasmid is first produced which is based on the vector pCYB2 from the IMPACT system (New England Biolabs). Via the multiple cloning site of pCYB2, with the aid of the restriction sites Ndel-Xmal (New England Biolabs), a DNA fragment produced via PCR amplification is cloned in according to standard methods and which codes for a variant of the VPl gene of mouse polyomavirus.
• a polyomavirus variant that has no cysteines in the sequence is used as the basis for this; the six cysteines of the wild-type protein were previously replaced with serine using standard mutagenization techniques.
• This variant of PyVPl has the advantage that the redox conditions of the solution have no influence on the state of the protein; this makes it easier to handle in many applications.
• a cysteine is later introduced into the inserted WW domain, a specific disulfide bridge between the WW domain and the proline-rich sequence can be carried out.
• Another variant uses a modification at point 249; the threonine present in the wild-type protein is replaced by cysteine.
• labeling with the aid of fluorescent dyes according to the prior art is advantageously possible.
• the protected localization in the pentamer allows marking at this point without undesirable side effects.
• the variant of polyoma virus VP1 used is correctly named PyVPl-CallS-T249C, hereinafter abbreviated as PyVPl.
• vplNImp (5 '-TAT ACA TAT GGC CCC CAA AAG AAA AAG C-3')
• vplCImp 5'-ATA TCC CGG GAG GAA ATA CAG TCT TTG TTT TTC C- 3 '.
• the C-terminal amino acids of the wild-type VP 1 protein from Gly383-Asn384 are simultaneously converted into Pro383-Gly384, since a C-terminally located asparagine is very unfavorable for the mtein cleavage system with regard to the cleavage properties.
• the point mutations mentioned do not affect the essential properties of the PyVP 1 protein.
• the t ⁇ c promoter of the pCYB2 vector delivers only small amounts of expression of the fusion protein, therefore the PyVPl-Intein-CBD fusion construct is isolated from the pCYB2 vector by means of a further PCR and converted into a highly expressing pET vector with 77 / ac promoter ( Plasmid pET21a, Novagen), cloned via Ndel-EcoRI restriction sites.
• the WW domain is cloned into the external loop of PyVPl between amino acid positions 148 and 149 in several steps.
• FBPl 1-WWaC 5'-ATA CTC TTC TAC CAC TAC CAT CAT CCG GCT TTT CCC AGG TAG ACT G-3 '
• FBPl 1 form-binding protein 11
• a WW domain is encoded in this gene sequence.
• the oligonucleotides simultaneously introduce a short linker sequence of 5 amino acids each, consisting of alternating glycine-serine amino acids.
• a second PCR on the vector described above amplifies the N-terminal fragment of PyVPl between amino acids 1 and 148 using the oligonucleotides vplNImp (see above) and vpl-150-WWaC (5'-ATA CTC TTC AGG TAG CGG CGT AAA CAC AAA AGG AAT TTC CAC TCC AG-3 ').
• a third PCR also amplifies the C-terminal fragment of PyVPl between amino acids 149 and the C-terminal end of the protein, using the oligonucleotides vpl-150-WWaN (5'-ATA CTC TTC AGC CGC TGC CTG TAT CTG TCG GTT TGT TGA ACC CAT G-3 ') and vplCImp (see above).
• a subsequent PCR with the oligonucleotides vplNImp and vplCImp now amplifies the ligation product of the three fragments, hereinafter abbreviated as PyVPl-WW150.
• the PCR product can then be cloned into the vector pCR-blunt (Invitrogen) using standard methods.
• the final cloning into the plasmid pET21a already described above can then be carried out.
• the last-created vector allows expression of the fusion protein (PyVP 1-WW 150) - intein-CBD with the help of the highly expressing 77 / "c promoter in E. coli BL21 (DE3) cells (Novagen).
• transformed cells are grown in 5 1 - Erlenmeyer flasks, each containing 2 1 LB medium, at 37 ° C until the OD 600 of the culture is 2.0 to 2.5. Protein expression is induced by 1 mM IPTG in the medium. The cultures are then incubated for a further 20 hours at 15 ° C .; the low temperature minimizes the cleavage of the intein fraction in the fusion protein under in v / v ⁇ conditions.
• the cells are harvested by centrifugation, dissolved in 70 ml resuspension buffer (20 mM HEPES, 1 mM EDTA, 100 mM NaCl, 5% (w / v) glycerol, pH 8.0), and disrupted by high pressure homogenization. After centrifuging the crude extract for 60 min at 48,000 g, a clear cell extract is obtained. This extract is applied at a flow rate of 0.5 ml / min at a temperature of 10 ° C on a 10 ml chitin affinity column (New England Biolabs).
• the column is then filled with 3 column volumes of the suspension buffer, 15 column volumes of a washing buffer of high ionic strength (20 mM HEPES, 1 mM EDTA, 2 M NaCl, 5% (w / v) glycerol, pH 8.0) and again 3 column volumes of the resuspension buffer; this removes all unwanted E. coli host proteins from the chitin matrix.
• a washing buffer of high ionic strength (20 mM HEPES, 1 mM EDTA, 2 M NaCl, 5% (w / v) glycerol, pH 8.0
• the cleavage of the PyVPl-WW150 monomer from the fusion protein by means of the self-splicing inactivity is induced by a pulse (3 column volumes) with 50 mM dithiothreitol (DTT), 50 mM hydroxylamine, or 30 mM DTT together with 30 mM hydroxylamine in the resuspension buffer.
• DTT dithiothreitol
• the loaded chitin matrix is incubated with one of the specified solutions for 14 hours at 10 ° C.
• the PyVP 1-WW 150 protein is released completely and can be separated from the chitin matrix and the other components of the fusion protein adhering to the matrix by standard column chromatography methods.
• a linear salt gradient with a concentration between 0.1 and 2.0 M NaCl is suitably used for this purpose.
• the chitin matrix is regenerated according to the manufacturer's instructions by washing the chitin material with 3 column volumes of an SDS-containing buffer (1% SDS (w / v) in resuspension buffer).
• the PyVPl-WW150 protein is expressed as a soluble pentamer in the process described and is native.
• 2a shows an SDS gel with the purified fractions of wild-type PyVPl (or the variant PyVPl-CallS-T249C derived therefrom) and the PyVPl-WW 150 variant, which has a higher mass due to the additional inserted amino acids.
• 2b shows comparative CD spectra of the proteins produced in 10 mM HEPES, 150 mM NaCl, pH 7.2, which show correct folding of the protein species.
• a deconvolution of the two CD spectra according to the prior art shows that in the case of the PyVPl-WW150 domain there is an increase in the ⁇ -sheet structure compared to the PyVPl protein. This indicates that the inserted WW domain has retained its native structure as a ⁇ -sheet.
• the example shows that, surprisingly, the WW domain, with correct folding, can be inserted into loop regions of protein structures under suitable conditions without significantly disrupting its native structure.
• the pyVPl-WW150 pentamer in solution contains the native WW domain inserted into the polypeptide chain and presents it when assembled on the outside of the virus-like envelope (see example 2)
• a protein (PyVP 1-WW 150) that has artificially inserted a WW domain can be produced using example 1.
• the binding properties of PyVPl-WW150 with respect to proline-rich ligands can be characterized by various methods. A cheap method uses the surface plasmon resonance; In the given example, the device Biacore X (from Biacore AB) is used. According to the manufacturer, a synthetic peptide of the sequence Cys-Ser-Gly-Pro-Pro-Pro-Pro-Pro-Pro-Pro-Pro-Leu-Pro is coupled to a sensor chip type CM5 via a thiol or amino coupling. First, a quantity of 80 resonance units (RU) of the specified peptide is immobilized on the surface. The subsequent measurements are each carried out at 25 ° C and a flow rate of 20 ⁇ l / min.
• RU resonance units
• Binding studies of PyVPl-WW150 on the sensor chip with immobilized proline-rich peptide are carried out under different solvent conditions.
• the first measurement is carried out under standard solvent conditions, with 10 mM HEPES, 1 mM EDTA, 150 mM NaCl, pH 7.2.
• the protein concentration of PyVPl-WW150 is varied in the range from 5 to 50 nM. It can be seen from FIG. 3a that PyVPl-WW150 binds to the sensor chip with high affinity. As expected, the bound amount is proportional to the protein concentration used.
• the binding constant K D of the PyVPl-WW150 protein is determined with a value of 5 nM (FIG. 3a).
• the binding is not permanent, but the protein dissociates after the sensor surface has been loaded in a slow process. This shows that the interaction between the interaction partners is reversible.
• Dulbecco's PBS (Gibco) is used as the solvent in the second measurement. The remaining experimental conditions are chosen analogously to the first experiment described above.
• Figure 3b demonstrates that the binding of the PyVPl-WW150 to Dulbecco's PBS shows no significant differences from the binding under standard conditions (Fig. 3a).
• the example shows that the changed solvent conditions have no significant influence on the The PyVPl-WW150 binds to the proline-rich peptide and suggests that the interaction between the two partners is stable even under physiological conditions, making the system usable in principle even under clinical conditions in the context of diagnostics or therapeutics.
• FIG. 3c shows that significant and specific binding of the PyVPl-WW150 protein to the sensor surface can also be observed under these conditions.
• the response signal on the sensor surface is proportional to the concentration of PyVPl-WW150 protein used. This shows that the interaction of the PyVP 1-WW 150 with the immobilized proline-rich peptide is independent of the presence of a mixture of other substances, such as those found in serum, for example.
• Another method for characterizing binding properties is a reversible immobilization of the WW domain on an inert matrix.
• a synthetic protein-rich peptide sequence Cys-Ser-Gly-Pro-Pro-Pro-Pro-Pro-Pro-Pro-Leu-Pro
• Sulfolink column material Pieris
• the PyVPl-WW150 protein purified according to Example 1 is applied to the column (solvent 10 mM HEPES, 1 mM EDTA, 150 mM NaCl, 5% glycerol, pH 7.2). As can be seen from FIG. 4, the protein binds to the matrix and appears only in small amounts in the wash fractions. Subsequent elution of the protein from the matrix is possible by adding 1% SDS or 300 mM arginine to the running buffer.
• the proline-rich peptide Cys-Ser-Gly-Pro-Pro-PiO-Pro-Pro-Pro-Pro-Leu-PiO is made with a fluorescein-maleimide derivative (from Molecular Probes) according to the manufacturer's instructions specifically on the N-terminal cysteine marked. After assembling the virus protein variants into capsids, a ten-fold molar excess of fluorescence-labeled peptide is added. Gel filtration (TSKGel G5000PWXL column, TosoHaas) clearly shows virus-like capsid shells and can be separated from free, unassembled capsid building blocks as well as from excess peptide and fluorescent dye.
• the peptide bound to the WW domains on the surface of the capsids elutes in the capsid fractions and can be detected by the specific absorption of the fluorescent dye (FIG. 5).
• FOG. 5 shows that the PyVPl-WW150 variant can form capsid structures (virus-like envelopes) under suitable conditions. These capsids are able to bind the proline-rich peptide. This allows molecular substances to be applied to the surface (outside) of the virus-like structures via the specific and strong interaction of WW domain and proline-rich sequence.
• Two different variants of the PyVPl protein are therefore produced which contain a WW domain at the amino terminus of the native wild-type protein (variant PyVPl-3C-WW1) or which carry the WW domain at an N-terminus shortened by 14 amino acids (Variant PyVPl-3C-WW [N-14]) and a variant of the PyVPl protein which carries a proline-rich sequence at the N-terminus (PyVPl- 3C [N-14] -Plp).
• the basis for these vainants is a PyVPl variant, which has the cysteines C19 and Cl 14 and into which a specific new cysteine is also introduced (analogous to the PyVPl-CallS-T249C variant). This variant is abbreviated as PyVPl-3C in the following.
• the WW domain is first amplified by means of PCR; the FBPl 1 gene of the mouse serves as a template analogous to example 1.
• 5'-AAT ATA TCA TAT GTC CAT CAT CCG GCT TTT CCC AGG TAG ACT-3 '(with Ndel interface), and 5'-TAT TAA TCA TAT GAG CGG CTG GAC AGA ACA TAA ATC are used as oligonucleotides for the PCR ACC TGA TGG-3 'used.
• the PCR product obtained is then cloned into the expression vector pET21a from Example 1, which contains the gene for a fusion protein PyVPl-Intein-CBD, via the interfaces Nde I - Nde I introduced by means of the oligonucleotides; At the 5 'end of the gene there is a singular Nde I interface.
• the expressed gene product of this vector is the desired protein PyVPl-3C-WWI.
• the cloning is carried out with a fragment of PyVPl -3 C shortened by 14 amino acids (PyVPl-3C-WW [N-14]), according to the standard method described in Example 1.
• a PCR is carried out on the PyVPl gene fragment, with 5'-GCG CGC GCA TAT GAG CAC CAA GGC TAG CCC AAG ACC CG-3 'and the oligonucleotide vplCImp (cf. Example 1).
• the resulting PCR product is digested with the restriction enzymes Nde I - Sma I and the fragment is cloned into the vector pET21a from Example 1 using standard methods. Expression and purification of the two proteins takes place in accordance with Example 1.
• the purified proteins are compared with the variants PyVPl and PyVPl-WW150 in FIG. 6a. It turns out that the proteins are soluble and can be produced natively.
• the change in the N-terminus due to the introduction of the WW domains has no significant negative influence on the assembly competence of the protein for the formation of virus-like envelope structures.
• GFP is a protein that shows green fluorescence in its native state (absorption maximum at 490 nm). It is ideal for marking complexes and associates.
• a PCR-based amplification of the GFP gene is first carried out, the plasmid being the template pEGFP-Nl (from Clontech) is used. At the same time, suitable restriction interfaces are introduced into the PCR product.
• the PCR is carried out using the oligonucleotides 5'-TTA TTT ACA TAT GGT GAG CAA GGG CGA GGA G-3 '(with Nde I interface), and 5'-ATA TCT TAA GTA CAG CTC GTC CAT GCC G-3' (with Afl Il interface).
• the PCR product thus obtained is cloned into the vector pTIP via the restriction sites and expressed there.
• This vector pTIP is a derivative of the Intein cleaning vector documented in Example 1 based on pET21a, with additionally introduced proline-rich sequences.
• the vector is constructed in such a way that a proline-rich sequence is fused at either the 5 'or 3' end of a gene inserted via a multiple cloning site.
• the proline-rich sequence mainly contains Pro-Pro-Pro-Pro-Pro-Pro-Pro-Leu-Pro.
• the GFP-PLP protein is produced and purified by means of chitin affinity chromatography, in accordance with the content of Example 1. The successful production and purification of GFP-PLP is documented in FIG. 6b.
• the GFP-PLP protein which carries the proline-rich sequence Pro-Pro-Pro-Pro-Pro-Pro-Pro-Leu-Pro at the C-terminus, can be produced in large quantities in a soluble manner.
• the glowing green color of the protein solution also shows that the protein can fold to its native structure.
• the Py-VPl-3C-PLP variant was produced analogously.
• PyVPl-3C- [N-14] was cloned into the vector pTIP in such a way that the proline-rich sequence contained in the vector was fused N-terminally to the Py-VPl-3C- [N-14].
• the two variants are incubated with proteins which contain proline-rich sequences.
• the PyVPl -3 C-WW1 protein is incubated with the previously prepared protein GFP-PLP (molar ratio 1: 6) for 10 min (10 mM HEPES, 1 mM EDTA, 150 mM NaCl, 5% glycerol, pH 7.2), and the capsid formation of the PyVPl variants induced by dialysis against a buffer containing 0.5 mM CaCl 2 (cf. Example 4).
• the successful detection of capsids FIG.
• the protein is able to bind the proline-rich peptide and, when assembled into a protein envelope, to bring the proline-rich peptide inside the capsid. This is shown in the gel filtration in FIG. 7b by the specific absorption of the fluorescent dye covalently bound to the peptide at 490 nm, which is found predominantly in the elution area of the capsids (9 to 10 ml).
• an analogous assembly attempt can be carried out using the variants of PyVPl-3C- [N-14] -PLP (proline-rich sequence at the N-terminus) and GFP-WW1 (WW domain at the N-terminus).
• PVPl-3C- [N-14] -PLP proline-rich sequence at the N-terminus
• GFP-WW1 WW domain at the N-terminus
• the experiments in Examples 5 and 6 show that variants of PyVPl with an N-tied-on fused WW domain are able to bind proline-rich sequences and, if appropriate, these and the molecular substances thereon when assembled to form capsids under suitable conditions To direct conditions into the interior of virus-like envelopes.
• the method described is therefore suitable for effecting a directed packaging of molecular substances in viruses or in virus-like capsids. It was also possible to show that variants of PyVPl with an N-terminally fused proline-rich sequence are able to bind WW domains and molecular substances attached to them.
• Point mutations which are carried out according to methods customary in the prior art, can be used to convert individual amino acids, which are not essential for the attachment of both adapter segments, into cysteines both in the WW domain and in the proline-rich sequences of the ligand.
• a disulfide bridge can then be specifically formed between the attached proline-rich ligand and the WW domain, each containing one or more cysteines. This bridging is decisively favored by the attachment of the WW domain and the ligand.
• the time-limited interaction between unlinked WW domain and proline-rich ligand lasts long enough to avoid the to form covalent bridging over the disulfide bridge.
• the interaction of the two adapter segments is thereby unlimited in time, since disulfide bridges are stable under physiological conditions, such as are present, for example, in the extracellular space.
• the disulfide bridge between WW domain and proline-rich ligand can be removed in vitro under reducing conditions (for example 50 mM DTT, DTE or ⁇ -mercaptoethanol); when the reducing agent is removed, bridging is also possible.
• an aspartate amino acid (position 8 in the WW domain) is converted into a cysteine by mutagenesis.
• the resulting cysteine-containing variant is named PyVP 1 - WW150-D8C in the following. Binding studies based on surface plasmon resonance show that this variant of the WW domain still contains the proline-rich ligand Cys-Ser-Gly-Pro-Pro-Pro-Pro-Pro-Pro-Pro-Pro-Leu- without the formation of the disulfide bridge. Pro binds. The extent of the interaction is, however, somewhat less than with PyVPl-WW150. This can apparently be attributed to the newly introduced cysteine.
• this addition can in turn be increased by adding 500 mM ammonium sulfate. This presumably intensifies the hydrophobic interactions between the proline-rich ligand and the WW domain. The strength of the interaction can thus be modulated by the ammonium sulfate concentration in the solvent.
• the disulfide bridge between the proline-rich ligand and the WW domain is then formed under slightly oxidizing conditions.
• a buffer is used for this, which contains both ammonium sulfate and maintains defined redox conditions.
• the latter conditions are achieved by using 1 mM GSSG and 5 mM GSH in the redox buffer (50 mM Tris, pH 8.5, 1 mM EDTA, 500 mM ammonium sulfate); Oxidized (GSSG) or reduced glutathione (GSH) acts as a redox shuffling system for the formation of the disulfide bridges (see R. Rudolph, In vitro folding of inclusion body proteins, FASEB J. 10, 49-56, 1996).
• the disulfide bridging is carried out at 15 ° C. for 24 h and terminated by dialysis against 50 mM Tris, 1 mM EDTA, pH 7. Under the latter conditions there is no longer any disulfide exchange; the disulfide bridges formed are stable.
• Two variants of a WW domain were used for bridging, in each of which an amino acid was exchanged for cysteine at one position. This is on the one hand the variant D8C, in which the aspartate at position 8 in the WW domain was exchanged for cysteine and, on the other hand, K19C, in which lysine 19 was replaced by cysteine.
• the molecular substance is a proline-rich peptide with the sequence CSGP S LP, which was labeled with a fluorescent dye (Oregon Green, OG, Molecular Probes) for the purpose of analysis at the amino group of the N-terminus.
• a fluorescent dye Opgon Green, OG, Molecular Probes
• the disulfide bridging was carried out as described in Example 7. To analyze the bridging, the sample was subjected to reversed-phase HPLC (HPLC column: YMC Protein-RP C 18 : running buffer A: 0.1% TFA in H2O, running buffer B: 80% ACN, 0.1% TFA).
• FIG. 9 (a) shows that this was the case for WW domain variant K19C, but not for D8C. This may be due to the steric inaccessibility of the D8C variant cysteine.
• Figure 9 (b) shows that the covalent interaction of the WW domain variant K19C and proline-rich peptide can be abolished by adding a reducing agent (50 mM DTT). The fluorescent peptide is then completely free again.
• Another field of application of the present invention is the separation of molecular substances from substance mixtures, as typically occurs in the purification of proteins from crude extracts (cell extracts).
• the affinity of the WW domain for proline-rich ligands is used to isolate proteins containing a WW domain from a complex mixture (crude extract) of proteins (principle of affinity chromatography).
• Example 3 A column according to Example 3 is used for this; the SulfoLink material (from Pierce, the reactivity of the matrix with SH groups is based on the iodoacetamide group at the end of a linker consisting of 10 CH 2 groups) is coupled to the peptide Cys-Ser-Gly- via a thiol coupling Load the Pro-Pro-Pro-Pro-Pro-Pro-Pro-Leu-Pro according to the manufacturer's instructions.
• SulfoLink material from Pierce, the reactivity of the matrix with SH groups is based on the iodoacetamide group at the end of a linker consisting of 10 CH 2 groups
• the peptide can also be coupled to other matrices, for example Aff ⁇ Gel 10 (from Biorad, the reactivity of the matrix with NH 2 groups is based on the N-hydroxysuccinimide group at the end of a linker, consisting of 10 CH 2 groups ) via the N-terminus of the peptide.
• the peptide coupling or the N-terminus of the peptide to a matrix based on CH-Sepharose 4B can take place (Sigma company, the reactive group of the matrix is also an N-hydroxysuccinimide ester). This also results in a covalent binding of the proline-rich ligand to a carrier material, which subsequently allows WW domain proteins to be purified.
• the PyVPl variant PyVPl-3C-WW1 from Example 5 (WW domain at the N-terminus of the PyVP 1 protein) is produced analogously to the information from Example 1 as a fusion protein with an intein and a chitin-binding domain ([PyVPl-3C -WWl] -intein-CBD). However, it is not purified by the standard method described using chitin affinity chromatography. Instead, the cell extract is applied to the column described above after cell disruption and subsequent centrifugation. 10 mM HEPES, 150 mM NaCl, 1 mM EDTA, 5% glycerol, pH 8.0 serves as the running buffer.
• the fusion protein is bound to the matrix and thus most of the other proteins of the cell extract are separated off in the course of or during the washing process.
• Elution with SDS then provides practically exclusively the complete WW domain-containing fusion protein, as well as proteolytic degradation products (which occur in the case of PyVPl in all comparable manufacturing processes according to the prior art) and molecular chaperones, which are known to bind directly to PyVP 1 assets and usually cannot be separated.
• proteolytic degradation products which occur in the case of PyVPl in all comparable manufacturing processes according to the prior art
• molecular chaperones which are known to bind directly to PyVP 1 assets and usually cannot be separated.
• this example shows that it is possible to use the system described to specifically separate molecules from a mixture of substances (crude extract) and thus purify them.
• the interaction of the adapter segments can also be used to produce bifunctional or bivalent hybrid molecules in vitro.
• two molecular substances are produced, which can have the same or different properties depending on the application, and which each carry one of the two adapter segments covalently linked.
• easily detectable dimers of the GFP protein are produced.
• a variant of GFP is produced analogously to the production of PyVPl with the help of the built-in expression ion system from Example 1 with a WW domain at the N-terminus of the GFP (GFP-WWl).
• a PCR is carried out on the vector pEGFP-NI (from Clontech, see Example 5) with the oligonucleotides 5'-TAT AGC TAG CGT GAG CAA GGG CGA GGA GCT GTT C-3 ⁇ and 5'-GGG AAT TAA GTA CAG CTC GTC CAT GCC G-3 '.
• the PCR product is ligated into the vector pET21a from example 5 via the interfaces Ne i - Sma I, which contains the fusion protein from intein and chitin binding domain (CBD) described in example 1 at the 3 'end of the insertion site.
• the WW domain which is described in Example 5, is located on the 5 ′ side of the insertion site.
• the plasmid encoding the fusion protein can be transformed into the strain E. coli BL21 (DE3).
• the fusion protein can then be prepared and purified analogously to Examples 1 and 3. With the method described, the protein GFP-WW1 can be produced in a purified form.
• a second variant of GFP is produced analogously with a proline-rich segment (Pro-Pro-Pro-Pro-Pro-Pro-Pro-Leu-Pro) at the C-terminus.
• the preparation and purification of the GFP-PLP protein is identical to the description of the protein in Example 5.
• the two GFP variants are then incubated together.
• the two proteins are connected to one another via the adapter segments and a GFP dimer results which can be distinguished from the GFP monomer by gel filtration on a TSK-PW2000XL gel filtration column (TosHaas).
• the example shows that the method described in the present invention can be used to connect any molecular substances which carry corresponding adapter segments based on WW domains or proline-rich peptide segments. This can result in homofunctional or heterofunctional associations.

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