Stabilized native protein complexes conjugated with branched molecules

Abstract

The present invention refers to a stabilized protein complex comprising at least two monomers. The native tertiary and / or quaternary structure of the protein complex is stabilized via crosslinkers having moieties B forming covalent linkages with reactive groups A of the monomer. The invention further relates to a method for producing the stabilized protein complex and monomer, respectively, and to crosslinkers used in this method.

Description

[0001] P132518PC00 Stabilized native protein complexes conjugated with branched molecules The present invention refers to a stabilized protein complex comprising a covalent bond between a crosslinker and reactive groups of two or more monomers of the protein complex, wherein the native tertiary and / or quaternary structure of the protein complex is stabilized, in particular under a denaturing condition. The present invention further relates to a method for the production of the protein complex, a crosslinker, and its use in producing the protein complex. The stabilized protein complex is characterized by reduced aggregation. Technical background Proteins are nature-derived molecules that perform many different tasks in organisms. Importantly, proteins often form homomeric or heteromeric complexes in order to function correctly. As enzymes for example proteins perform highly specific catalytic chemical transformations. As ligands and receptors, they bind their different partners with high specificity. These features are also important when using proteins in biotechnological and biomedical applications including diagnostics and therapeutics. A major bottleneck for the use of proteins in such applications is the loss of protein structure integrity due to the often required unphysiological conditions (e.g., elevated temperatures, altered buffer conditions, supplemented additives, incl. chaotropic agents, detergents, organic solvents). The specific three-dimensional structure is what gives proteins their specific functions. The “tertiary structure” of a protein complex refers to the three-dimensional shape of a protein monomer. The “quaternary structure” of a protein complex refers to the association of monomers into closely associated assemblies. Each of the monomers is for example an amino acid chain and has its own primary, secondary, and tertiary structure, respectively, and the monomers associate into a protein complex with its own quaternary structure. If the structure is disrupted, the protein does not function anymore and becomes useless for application. Consequently, the stabilization of protein structures is a central aspect in the development of suitable proteins including enzymes and receptors for biotechnological and biomedical applications. The complexity of interactions in protein tertiary and / or quaternary structures and the sensitivity of proteins including enzymatic activity, receptor recognition on sequence alterations, render efforts towards protein including enzyme stabilization very challenging. A minimal invasive strategy involves the use of covalent protein modifications (e.g., pegylation or glycosylation) being mainly applied to increase biostability for therapeutic applications. Alternatively, protein stabilization can be achieved via alterations in the protein sequence applying directed evolution, consensus-based mutagenesis or computational approaches which can be complimented by the introduction of non-proteinogenic amino acids. These approaches aim for improved protein core interactions, structure rigidification, and / or surface charge distribution and often require multiple rounds of optimization to achieve relevant stabilization effects. Pelay-Gimeno, M.; Bange, T.; Hennig, S.; Grossmann, T.N., Angew. Chem. Int. Ed. 2018, 57, 11164; Neubacher, S.; Saya, J.M.; Amore, A.; Grossmann, T.N., J. Org. Chem. 202085 (3), 1476-1483 describe the stabilization of protein monomers through the use of trivalent crosslinkers. WO2018197893A refers to trivalent crosslinkers for the production of de novo, small, peptide structures. Notably, the use of afore mentioned approaches for the stabilization of protein complexes is largely unexplored. While inter-protein crosslinking has been used for probing proximity (dos Santos, A.P.A.; da Silva, J.K.; Neri, J.M.; Neves, A.C.O.; de Lima, D.F. et al., Org. Biomol. Chem., 2020, 18, 9398-9427), crosslinking for quaternary structure stabilization has not been widely performed, examples involve the introduction of disulfide bridges (Robinson, C.R.; Sauer, R.T. Biochem., 2000, 39, 12494-12502; Meinhold, D.; Beach, M.; Shao, Y.; Osuna, R.; Colón, W. Biochem., 2006, 45, 9767-9777) or the fusion of DNA- binding homo-dimers (Liang, H.; Sandberg, W.S.; Terwilliger, T.C. Proc. Natl. Acad. Sci. USA, 1993, 90, 7010-7014, Akanuma, S.; Matsuba, T.; Ueno, E.; Umeda, N.; Yamagishi, A. J. Biochem., 2010, 147, 371-379; Dellarole, M.; Sánchez, I.E.; Freire, E.; de Prat-Gay, G. Biochem., 2007, 46, 12441-12450). The introduction of reactive non-natural amino acids has been used to probe for protein complexes (Shu, X.; Asghar, S.; Yang, F.; Li, S- T.; Wu, H. et al., Front. Chem., 2022, 10, 1-11.) but also to trap known transient or weak dimeric complexes (Cigler, M.; Müller, T.G.; Horn-Ghetko, D.; von Wrisberg, M-K.; Fottner, M. et al., Angew. Chem. Int. Ed., 2017, 56, 15737-15741; Zhou, L.; Chai, F.; He, Y.; Zhou Z.; Guo, S. et al., Commun. Biol., 2022, 5, 1-12). Thus, there exists an urgent need for stable protein complexes ensuring long-lasting protein function while minimizing undesired side effects. Protein complexes of the present invention have a similar overall structure and function to their unmodified, i.e., native protein counterparts. This reduces alterations of their function (minimization of negative side effects) while increasing their overall stability (e.g., under harsh and / or storage conditions). These protein complexes have a stabilized tertiary and / or quaternary structure, which may also include the stabilization of a particular conformation, if there are different native states, i.e., conformations possible to either hamper or enhance a certain structure and hence its associated function. Overall, the aim is to retain a particular tertiary and / or quaternary structure within a protein complex when compared to the unmodified corresponding native protein complex, in particular under stressful conditions such as elevated temperatures, altered buffer conditions, supplemented additives, incl. chaotropic agents, detergents, and organic solvents, respectively. Thus, protein complexes of the present invention are useful for a variety of already existing applications on the one hand and novel applications on the other. Hence, applications that already use related proteins can be made more efficient, faster and / or easy to use. Novel applications that were hampered by the lack of stable protein complexes can be implemented based on the present invention. Summary of the invention The present invention refers to a protein complex comprising or consisting of two or more monomers in native tertiary and / or quaternary structure which is fixed by a crosslinker having a core structure Q and at least three moieties B covalently linked to reactive groups A in one or more of the monomers forming a linkage F, wherein group A is independently a sulfhydryl group or a bioorthogonal group, and moiety B is independently a sulfhydryl-reactive group or a bioorthogonal group. In the protein complex A is a sulfhydryl group and B is a sulfhydryl-reactive group, or wherein A is a bioorthogonal group and B is a bioorthogonal group. The linkage F is for example selected from the group consisting of , wherein R is an alkyl, R1is independently selected from H, F, -O(CH2)(CO)OH, -O(CH2)-pBzOH, R2is independently selected from H, -CH3, -(C6H6)CH2COOH, -pyridyl, and R3is independently selected from H, -CH3. Each moiety B of the crosslinker is for example covalently linked to a different reactive group A of a different monomer, or all of the at least three moieties (B) of a crosslinker are covalently linked to three or more reactive groups (A) of one monomer, or one or more of the at least three moieties (B) of the crosslinker are covalently linked to one or more reactive groups (A) of one monomer, and one or more of the at least three moieties (B) are covalently linked to one or more reactive groups (A) of one or more other monomers, or combinations thereof. The protein complex is for example multi-stabilized comprising two or more monomers covalently linked to two or more crosslinkers. The protein complex comprises or consists for example of at least two monomers, of at least three monomers, or of at least four monomers. Q is for example selected from the group consisting of , or a combination thereof, wherein n is an integer of 1, 2, 3, 4 or 5, wherein a dashed line indicates a site where Q is directly bound to B or optionally via L. L is for example selected from the group consisting of wherein X is selected from NH or O, Y is selected from NH or O, Z is selected from NH or O, m is an integer of 1, 2, 3, 4 or 5, p is an integer of 1, 2, 3, 4 or 5, o is an interger in the range of 1, 2, 3 or 4, wherein the dashed line indicates a site where L is bound to Q or B. B is for example selected from the group consisting of wherein Z is selected from F, Cl, Br, I, Tos (O−SO2−C6H4−CH3), and Mes (O−SO2−CH3), R1is selected from H, F, -O(CH2)(CO)OH, -O(CH2)-pBzOH, R2is selected from H, -CH3, -(C6H6)CH2COOH, -pyridyl, R3is selected from H, -CH3, wherein each dashed indicates a site where B is bound to the linker L. In a protein complex of the present invention Q is selected from the group consisting of wherein n is an integer of 1, 2, 3, 4 or 5, wherein each dashed line in Q indicats a site where Q is directly bound to B or optionally via L; L is optionally selected from the group consisting of , or a combination thereofwherein X is selected from NH or O, Y is selected from NH or O, Z is selectedfrom NH or O, m is an integer of 1, 2, 3, 4 or 5, p is an integer of 1, 2, 3, 4 or 5, o is an interger in the range of 1, 2, 3 or 4, wherein the dashed line indicates a site where L is bound to Q or B; and B is selected from the group consisting of combination thereof, wherein Z is selected from F, Cl, Br, I, Tos (O−SO2−C6H4−CH3), and Mes (O−SO2−CH3), wherein each dashed line in B indicates a site where B is optionally bound to L or directly to Q. A protein complex of the present invention is for example an enzyme, a cytokine, a chemokine, a growth factor, a hormone, an antibody, a receptor, an antigen, structural protein, a defensive protein, a storage protein, a transport protein, a contractile, a signaling, a membrane, a secreted protein or combinations thereof. The present invention further refers to a method for preparing a protein complex of the present invention comprising or consisting of the steps: a) providing a protein complex in its tertiary and / or quaternary structure, wherein one monomer comprises at least three cysteine sulfhydryl groups A and / or bioorthogonal groups A, b) optionally performing a reaction with a connector to add a bioorthogonal group to one or more of the at least three cysteine residues of the monomer, and c) contacting the monomer with a crosslinker comprising a core Q bound to at least three reactive moieties (B) such that each moiety forms a linkage (F) with one of the at least three cysteine residues or bioorthogonal group(s) of the monomer; a) providing a protein complex in its tertiary and / or quaternary structure, wherein at least three monomers comprise a sulfhydryl group A and / or a bioorthogonal group A, b) optionally performing a reaction with a connector to add a bioorthogonal group to one or more of the at least three cysteine residues of the monomer and c) contacting the at least three monomers with a crosslinker comprising a core Q bound to at least three moieties (B) such that each moiety forms a linkage (F) with the cysteine residue or bioorthogonal group of each of the at least three monomers; a) providing a protein complex in its tertiary and / or quaternary structure, wherein at least two monomers, each comprise at least one cysteine sulfhydryl group A and / or bioorthogonal group A, b) optionally performing a reaction with a connector to add a bioorthogonal group to one or more of the at least three cysteine residues of the monomers, and c) contacting the at least two monomers with a crosslinker comprising a core Q bound to at least three moieties (B) such that each moiety forms a linkage (F) with the cysteine residue or bioorthogonal group of each of the at least two monomers. The method further comprises combinations of these methods. A connector comprises or consists for example of a bioorthogonal group A´, a component L and a moiety B reacting with the reactive group A of the monomer. A sulfhydryl group A is for example introduced in the monomer which reacts with an electrophilic group of the connector comprising the bioorthogonal group to introduce the bioorthogonal group A in the monomer. The sulfhydryl-reactive moiety B of the crosslinker forms for example a covalent linkage with the sulfhydryl group A of the protein complex, or the bioorthogonal moiety B of the crosslinker forms a covalent linkage with the bioorthogonal group A of the protein complex, or a combination thereof in one or more monomers of the protein complex. All documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. Description of the figures Fig. 1 shows exemplary reaction schemes for forming protein complexes of the present invention. It is not limited to monomer, dimer or trimer but can be extended to tetramers, pentamers, hexamers, and higher multimers. P1, P2, P3 can be part of a higher-order complex, i.e. a dimer could be part of a tetramer or multimer. Group A can be a cysteine residue, an amino acid residue that has been modified so it bears an alkyn, azide or other bioorthogonal group, or a non-natural amino acid bearing an alkyne, azide or other bioorthogonal moiety. Group A reactive groups that react with moieties to give linkage F can be in a single, in two or three different monomers and a combination thereof. Q is a core structure that carries three moieties LB. B is an electrophile, alkyne, azide or other bioorthogonal moiety. Reactivities of A and B are matched accordingly, e.g. if A=cysteine sulfhydryl, then B=electrophile; if A=alkyne, then B=azide. Fig. 2 shows the one-component method (top) where sulfhydryl cysteines (alternatively a present bioorthogonal group) A on the protein are directly modified with a three-armed electrophilic (alternatively a corresponding bioorthogonal moiety) crosslinker Q(LB)3. Reactive groups A on the protein react with moiety B on the crosslinker to give linker units F (top left). Q(LB)3 can be Ta4, where each iodoacetamide can react with a group A on the protein complex which can be cysteine sulfhydryl groups to give a thioether bearing linkage F (top right). The two-component method (bottom) introduces a different reactive group A before crosslinking with Q(LB)3. Cysteine sulfhydryl groups A on the protein are reacted with connectors A’-L-B, where B is a sulfhydryl-reactive moiety and A’ is a bioorthogonal moiety. In a second step, the biorthogonal groups (A) are crosslinked via a three-armed crosslinker Q(LB)3 (bottom left). The connector A’-L-B can be 2-iodo-N-(prop- 2-yn-1-yl)acetamide that reacts with cysteine sulfhydryl groups (A) on the protein complex. The crosslinker Q(LB)3 can be Ta-azide which reacts with the previously introduces alkyne groups (A) on the protein complex in a second step (bottom right). Fig. 3A shows protein complexes wherein all moieties B of a crosslinker are covalently linked to three or more reactive groups A of one monomer wherein optionally multiple crosslinker and additional monomers (Px) are present. Fig. 3B depicts protein complexes wherein one or more moieties B of the crosslinker are covalently linked to one or more reactive groups A of one monomer and one or more moieties B are covalently linked to one or more reactive groups A of one or more other monomers, wherein optionally multiple crosslinker and additional monomers (Px) are present. Fig. 3C shows protein complexes wherein each moiety B of the crosslinker is covalently linked to a reactive group A of different monomers, wherein optionally multiple crosslinker and additional monomers (Px) are present. A protein complex can combine 3A, 3B, and 3C. Fig. 4 shows A) crystal structure of trimer Pseudomonas fluorescens aryl esterase (PDB ID: 1va4) including designed modification sites (T3 and Q174) for crosslinking. B) Denaturing SDS PAGE of PFE variants with and without crosslinking confirming covalent linkage of the native trimer. C) Mass spectra (ESI-TOF) of crosslinked protein trimers including charge state of most abundant signal. D) Thermal unfolding curves of indicated variants using nanoDSF, with indicated TM values. E) Relative initial rates of esterase activity for PFE-wt and the three covalent trimers (relative to initial rate of PFE-DM-Ta2 at the given GuHCl concentration). Fig. 5 depicts A) SDS PAGE of PFE-Q173 where Q is unmodified PFE-Q173, Q3 is IA-C3 modified PFE-Q173, Q5 is IA-C5 modified PFE-Q173, Q3B and Q5B are Q3 and Q5, respectively reacted with Bz-azide (B), and Q3T and Q5T are Q3 and Q5 reacted Ta-azide (T). B) Mass spectra (ESI) of unmodified (left) and IA-C3 modified (right) PFE-Q173. C) Initial rates of esterase activity of two-component crosslinked PFE-Q173-IA3-Ta, PFE-T2- IA3-Ta and PFE-DM-IA3-Ta2. Protein samples were pre-treated at indicated temperatures for 1h. Fig. 6 depicts A) Crystal structure of trimer Rhodanese domain-containing protein from Lactiplantibacillus plantarum (PDB ID: 3fnj) including one of two designed modification sites for crosslinking. B) SDS PAGE of unmodified I4 and covalent trimer I43Ta2. C) Mass spectra (ESI) of crosslinked I43Ta2 trimer. D) Thermal unfolding curves of I4 and I43Ta2 using nanoDSF, with indicated ^TM values. Fig.7 shows A) Crystal structure of trimer Enoyl-CoA hydratase / isomerase family protein from Hyphomonas neptunium (PDB ID: 5c9g) including one of two designed modification sites for crosslinking. B) SDS PAGE of unmodified e4 and covalent trimer e43Ta2. C) Mass spectra (ESI) of crosslinked e43Ta2 trimer. D) Thermal unfolding curves of e4 and e43Ta2 using nanoDSF, with indicated ^TM values. Fig.8 shows A) Crystal structure of trimer Tautomerase_3 domain-containing protein (a4) from Neosartorya fumigate (PDB ID: 3c6v) including one of two designed modification sites for crosslinking. B) SDS PAGE of unmodified a4 and covalent trimer a43Ta2. C) Mass spectra (ESI) of crosslinked a43Ta2 trimer. D) Thermal unfolding curves of a4 and a43Ta2 using nanoDSF, with indicated ^TM values. Fig.9 shows A) Crystal structure of trimer Tautomerase_3 domain-containing protein (a4) from Neosartorya fumigate (PDB ID: 1vmf) including one of two designed modification sites for crosslinking. B) SDS PAGE of unmodified b4 and covalent trimer b43Ta2. C) Mass spectra (ESI) of crosslinked b43Ta2 trimer. D) Thermal unfolding curves of b4 and b43Ta2 using nanoDSF, with indicated ^TM values. Fig.10 depicts A) Crystal structure of tetramer asparaginase II from E. coli (PDB ID: 3eca) including designed modification sites for crosslinking. B) SDS PAGE of EcAsp-wt and covalent dimer EcAsp-1-Ta2. C) Mass spectra (ESI) of crosslinked EcAsp-1-Ta2 dimer. D) Thermal unfolding curves of EcAsp-wt and EcAsp-1-Ta2 using nanoDSF. E) Analytical size-exclusion chromatography of EcAsp-1 and EcAsp1-Ta2 (eluent: 150 mM sodium phosphate pH 7.0, column: Agilent Bio SEC-3, injections: 4 pmol). F) Asparaginase activity performed with Asparaginase enzyme activity assay kit (Sigma) according to manufacturer’s protocol showing similar activities of EcAsp-wt and EcAsp-1-Ta2. Fig.11 shows A) Crystal structure of tetramer Borneol dehydrogenase of Pseudomonas sp. TCU-HL1 (PDB ID: 6m5n) including designed modification sites for crosslinking. B) Thermal unfolding curves of BDH-wt and BDH-X2-Ta2 using nanoDSF, with indicated Ti and ^Ti values. C) Mass spectra (ESI) of crosslinked BDH-X2-Ta2 dimer. D) SDS PAGE of unmodified BDH-X2 and covalent dimer BDH-X2-Ta2. E) Enzymatic activity after 15-min incubation at temperature T. Fig. 12 depicts A) Crystal structure of dimer human galectin-1 (PDB ID: 1gzw)) including designed modification sites for monomer crosslinking of Gal1-1. B) Mass spectra (ESI) of crosslinked Gal1-1-Ta monomer. C) Thermal unfolding curves of Gal1-wt (C), non- crosslinked Gal1-1 (1) and Gal1-1-Ta (1-Ta) using nanoDSF. D) Crystal structure of dimer human galectin-1 (PDB ID: 1gzw)) including designed modification sites for dimer crosslinking of Gal1-3. E) Mass spectra (ESI) of crosslinked Gal1-3-Ta2 dimer. F) Thermal unfolding curves of Gal1-wt (C), non-crosslinked Gal1-3 (3) and Gal1-3-Ta (3-Ta2) using nanoDSF. G) SDS PAGE of unmodified Gal1 (C), non-crosslinked Gal1-1 (1) and Ga1-3 (3), crosslinked Gal1-1 monomer (1-Ta) and crosslinked Ga1-3 dimer (3-Ta2). Fig. 13 shows long-term temperature resistance of PFE-DM-Ta2. Fig. 14 depicts prolonged activity of EcAsp-1-Ta2. (A) Residual activity after pre- incubation of enzymes in PBS pH 7.4 for 5 min at 25 or 65 °C. (B) Activity after storage in PBS pH 7.4 for indicated times. Fig. 15 shows Ta4 crosslinked PFE (PFE-DM-Ta2) resisting aggregation under thermal stress. (A) Calculated size distributions of PFE-wt and PFE-DM-Ta2 particles at given temperatures between 40 to 69°C, determined by dynamic light scattering (DLS). (B) Crystal structure of PFE (PDB = 1va4) showing expected radii of a trimeric complex. (C) Cumulant radius analysis of PFE DLS data shows predominant particle radius between 20 to 90°C, demonstrating aggregation resistance of PFE-DM-Ta2. Fig. 16 shows Gal1-3-Ta2 dimer resisting aggregation. (A) Turbidity analysis using back- reflection shows aggregation of Gal1-wt and lack of aggregation for Gal1-3-Ta2. (B) HPLC- SEC in PBS pH 7.4 shows a mixture of monomer and dimer states for Gal1-wt and only the dimer state for Gal1-3-Ta2. Fig. 17 depicts stabilized protein complexes resisting aggregation. Turbidity analysis using back-reflection shows aggregation onset at higher temperatures for stabilized protein complexes compared to unmodified wt. (A) PFE wt and PFE-DM-Ta2. (B) BDH-wt and BDH-X22-Ta2. (C) EcAsp-wt and EcAsp-1-Ta2. Detailed description The present invention refers to protein complexes comprising or consisting of at least two monomers, wherein the monomers are identical (homomer) or different (heteromer). The protein complex comprises or consists for example of 2 to 60, 3 to 55, 4 to 50, 5 to 45, 6 to 40, 7 to 35, 8 to 30, 9 to 25 or 10 to 20 monomers, i.e., the protein complex is for example a dimer, a trimer, tetramer, pentamer, hexamer, heptamer, for example up to a 60-mer. Preferably the protein complex is a dimer, trimer or tetramer. The protein complex or the monomer in its native state is conjugated with branched molecules, preferably a cross-linker. The native state is one of the originally existing conformations and / or assembles adopted by a monomer and / or the protein complex. The branched molecule is for example a crosslinker (Q(LB)x). The crosslinker comprises or consists of three components which are Q, optionally L, and B. x of the crosslinker is for example at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 at least 10 or for example 3 to 10, 2 to 9, 4 to 8, 5 to 6 or 3, 4, 5, 6, 7, 8, 9, or 10, preferably 3 moieties B and optionally L. Each B is bound to core Q optionally via linker L. The crosslinker comprises at least three moieties (B) to react with at least one monomer of at least two monomers of a protein complex. The different moieties (B) of the branched molecule, such as a crosslinker (Q(LB)x), react with different reactive groups (A) of one monomer or with different groups (A) of different monomers to form linkage F. Linkage F is formed by the reaction of the reactive group A of the monomer and the moiety B of the crosslinker, which form for example a covalent bond. The crosslinked sites are for example covalent bonds (F). The crosslinked sites are of the same or of different protein monomers. Different crosslinking modes of the monomers according to the present invention are shown for example in Fig. 1. The crosslinker (Q(LB)x) tethers for example at least three different groups (A) of one monomer together or at least three monomers via one group (A) in each of the monomers. The crosslinker (Q(LB)x), optionally comprising component L, has at least three moieties (B) that react with a monomer, i.e., one or more monomers. The linker moiety (B) reacts preferably with a reactive group (A) of the monomer. The reactive groups (A) such as sulfhydryl groups (-SH) of a cysteine residue and / or bioorthogonal groups on the monomer are present naturally or are inserted through protein engineering or other known methods. The present invention refers to branched molecules such as crosslinkers (Q(LB)x) selected from the group consisting of 1,1',1''-(1,7,13-trioxa-4,10,16-triazacyclooctadecane- 4,10,16-triyl)tris(2-chloroethan-1-one), 4,10,16-tris(vinylsulfonyl)-1,7,13-trioxa-4,10,16- triazacyclooctadecane), 1,1',1''-(1,4,7-triazonane-1,4,7-triyl)tris(2-chloroethan-1-one), 1,4,7-tris(vinylsulfonyl)-1,4,7-triazonane), N,N',N''-(benzene-1,3,5- triyltris(methylene))tris(2-chloroacetamide), N,N',N''-(benzene-1,3,5- triyltris(methylene))tris(2-iodoacetamide), N,N′,N′′-((1,3,5-triazinane-1,3,5-triyl)tris(2- oxoethane-2,1-diyl))tris(2-chloroacetamide), N,N',N''-((1,3,5-triazinane-1,3,5-triyl)tris(2- oxoethane-2,1-diyl))tris(2-iodoacetamide), N,N',N''-((1,3,5-triazinane-1,3,5-triyl)tris(2- oxoethane-2,1-diyl))tris(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide), N,N',N''- (benzene-1,3,5-triyltris(methylene))tris(3-(2-chloroacetamido)propanamide), N,N',N''- (benzene-1,3,5-triyltris(methylene))tris(3-(2-iodoacetamido)propanamide), N,N',N''- (benzene-1,3,5-triyltris(methylene))tris(3-(2-(2-chloroacetamido)ethoxy)propanamide), N,N',N''-(benzene-1,3,5-triyltris(methylene))tris(3-(2-(2- iodoacetamido)ethoxy)propanamide), N,N',N''-((1,3,5-triazinane-1,3,5-triyl)tris(2- oxoethane-2,1-diyl))tris(3-(2-chloroacetamido)propanamide), N,N',N''-((1,3,5-triazinane- 1,3,5-triyl)tris(2-oxoethane-2,1-diyl))tris(3-(2-iodoacetamido)propanamide), N,N',N''- ((1,3,5-triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))tris(3-(2-(2- chloroacetamido)ethoxy)propanamide), N,N',N''-((1,3,5-triazinane-1,3,5-triyl)tris(2- oxoethane-2,1-diyl))tris(3-(2-(2-iodoacetamido)ethoxy)-propanamide), N1,N1',N1''-((1,3,5- triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))tris(N6,N6-dicyclobenzoazacyclo- octynylamide), N1,N1',N1''-(nitrilotris(propane-3,1-diyl))tris(N6,N6- dicyclobenzoazacyclooctynylamide), and a combination thereof. Alternatively the crosslinker is selected from the group consisting of N,N′,N′′-((1,3,5-triazinane-1,3,5- triyl)tris(2-oxoethane-2,1-diyl))tris(2-chloroacetamide), N,N',N''-((1,3,5-triazinane-1,3,5- triyl)tris(2-oxoethane-2,1-diyl))tris(2-iodoacetamide), N,N',N''-((1,3,5-triazinane-1,3,5- triyl)tris(2-oxoethane-2,1-diyl))tris(3-(2-(2-chloroacetamido)ethoxy)propanamide)), N,N',N''-((1,3,5-triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))tris(3-(2-(2- iodoacetamido)ethoxy)propanamide)), N,N',N''-(benzene-1,3,5-triyltris(methylene))tris(3- (2-(2-chloroacetamido)ethoxy)propanamide), N,N′,N′′-(benzene-1,3,5- triyltris(methylene))tris(2-chloroacetamide), N,N',N''-(benzene-1,3,5- triyltris(methylene))tris(2-iodoacetamide), N,N',N''-(benzene-1,3,5- triyltris(methylene))tris(3-(2-(2-chloroacetamido)ethoxy)propanamide), N,N',N''- (benzene-1,3,5-triyltris(methylene))tris(3-(2-(2-iodoacetamido)ethoxy)propanamide), or a combination thereof. Preferred crosslinker is a trivalent crosslinker comprising three reactive moieties (B). The protein complex comprises or consists for example of an enzyme such as Pseudomonas fluorescens aryl esterase (PFE, trimer), where subunits PFE-Tx, PFE-Qx and PFE-DMx, respectively, stabilized according to the present invention are functional at 1.5 M guanidinium hydrochloride compared to an unmodified PFE. Further examples of protein complexes are enoyl-CoA hydratase / isomerase family protein of Hyphomonas neptunium (trimer), rhodanese domain-containing protein of Lactiplantibacillus plantarum (trimer), tautomerase 3 domain-containing protein of Neosartorya fumigate (trimer), D-beta-hydroxybutyrate dehydrogenase (e.g., BH3498 protein) of Halalkalibacterium halodurans (trimer), asparaginase II of E. coli (tetramer), borneol dehydrogenase of Pseudomonas sp. TCU-HL1 (tetramer) or human galectin-1 (dimer). The present invention provides methods for producing stabilized protein complexes via crosslinking. The protein complex may have any function, e.g., enzyme, cytokine, chemokine, growth factor, hormone, antibody, receptor, antigen function, structural protein, a defensive protein, a storage protein, a transport protein, a contractile, a signaling, a membrane, a secreted protein etc. or combinations thereof. A protein may even have a combination of two or more functions which is for example a bifunctional or multifunctional protein. In some embodiments, the protein is an enzyme. As is apparent to a skilled person, the crosslinked monomers described herein form protein complexes composed of more than one polypeptide or peptide chain (monomer) representing the protein complex. The monomer(s) of the protein complex is selected from the group consisting of a hormonal protein, an enzymatic protein such as esterase PFE, a structural protein, a defensive protein, a storage protein, a transport protein, a receptor protein, a contractile, a signaling, a membrane, a secreted protein or a combination thereof. A protein may even have a combination of two or more functions which is for example a bifunctional or multifunctional protein. The reactive groups (A) such as sulfhydryl groups of a cysteine residues are present in the different monomers such as polypeptides and / or peptide chains naturally or are introduced via known molecular methods such as modified amino acids comprising a cysteine residue and / or a bioorthogonal group. For example, the present invention refers to monomers and protein complexes, respectively, having increased stability, and methods for increasing the stability of for example a homodimeric, homotrimeric or homotetrameric protein. As an example of one of the many embodiments described herein a homodimer ligand-binding protein is provided having three sulfhydryl groups (A) per monomer. The three sulfhydryl groups (A) are spatially arranged such that two cysteine residues of one monomer and one cysteine residue of the other monomer are in spatial proximity forming a triangle. The cysteine triangle is for example contacted with a trivalent sulfhydryl-reactive crosslinker (Q(LB)x) forming covalent bonds with each of the three cysteine residues (A) and between the monomers of the dimer. Further examples of protein complexes comprising or consisting of crosslinked monomers according to the present invention are for example shown in Fig.1 for dimers and trimers. In particular, the protein complex and the methods for its production of the present invention improve the stability of the monomer´s and / or protein complex´s native complex assembly. As is clear to a skilled person, an increase in stability refers to an increase in stability of the crosslinked protein complex as compared to the stability of the protein complex without crosslinking. The term “increased stability” as used herein, refers to an increase in resistance to or a decrease in susceptibility to denaturation. Denaturation refers to the loss of secondary, tertiary and / or quaternary structure and of the biological function. For example, enzymatic activity of most proteins is reduced or lost when denatured. Denaturation can occur as a result of mechanical agitation, radiation, increased temperature, or by chemical denaturants. Improved stability according to the present invention refers for example to the presence of a higher ratio of folded to unfolded protein when crosslinked, relative to that of the protein without crosslinking improved stability can be determined by examining the amount of folded protein present under varying conditions, e.g., temperature, detergent, denaturing agents, and pH. The protein complex and the methods of its production of the present invention improve for example the thermal stability (e.g., shown in Fig. 4 to Fig. 14) and / or stability against chemical denaturants (chemical stability) and / or proteases. The stability of the protein complex can be for example determined by measuring the Tm. The term “Tm” refers to the temperature at which 50% of the monomer and / or protein complex has unfolded. Typically, the higher the Tm, the more stable the monomer and / or the protein complex. In some embodiments the methods are for increasing the Tm of a monomer and / or protein complex. In some embodiments, the stability of the monomer and / or protein complex can be determined by measuring the effects of chemical agents on the monomer and / or protein complex. Chemical denaturants are agents that can disrupt non-covalent interactions and covalent bonds within a protein. Exemplary chemical denaturants include guanidinium hydrochloride, guanidinium thiocyanate, urea, organic solvents, salts, reducing agents (e.g. dithiothreitol, beta-mercaptoethanol, dinitrothiobenzene), detergents, and acids. Native monomer or native protein complex means that the monomer or the protein complex is adopting a tertiary and / or quaternary structure comprising variances which are also present in the native environment. A native monomer or native protein complex is at least partially preformed and for example coexists with other conformational or assembly states. This preformed, already existing tertiary and / or quaternary structure is stabilized according to the present invention. The branched molecules are not used to crosslink completely artificial assemblies that do not occur under native conditions. The native monomer or native protein complex is a natural or an artificial monomer or protein complex, or a combination thereof. The native monomer or native protein complex includes one or more natural and one or more artificial monomers. In the following, the elements of the present invention will be described in more detail. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The various described examples and embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise. Throughout this specification and the claims, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps. The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as", “for example”), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention. Protein complexes of the present invention are for example homomultimers or heteromultimers comprising or consisting of the same monomer or different monomers. The protein complex comprises or consists of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten monomers. The protein complex comprises or consists of at least two monomers. It comprises or consists of at least three monomers. It comprises or consists of at least four monomers. The protein complex comprises or consists of for example 2 to 60, 3 to 55, 4 to 50, 5 to 45, 6 to 40, 7 to 35, 8 to 30, 9 to 25 or 10 to 20 monomers. The protein complex comprises or consists of for example of 2, 3, 4, 5, 6, 7, 8, 9 or 10 monomers; preferably the number of monomers is 2, 3, 4, 5, or 6. The monomer in a protein complex of the present invention reacts with the branched molecule such as a crosslinker in different ways. The crosslinker (Q(LB)x) comprises or consists of moieties (B) which react with reactive groups (A) of the monomer; the linker component (L) is optional. A crosslinker comprises three or more moieties (B) to react with reactive groups (A) of monomers. The crosslinker comprises or consists of or has for example 2, 3, 4, 5, 6, 7, 8, 9, 10 moieties (B); preferably the crosslinker comprises or consists of 3 moieties (B). The moieties (B) of the crosslinker (Q(LB)x), wherein the linker component (L) is optional, react with the reactive groups (A) of the monomer(s) (Px) for example in the following ways: 1) each B of the crosslinker reacts with A of a different monomer (Px); 2) all B of the crosslinker react with three or more A of only one monomer; 3) one or more B of the crosslinker react with one or more A of one monomer and one or more B of the crosslinker react with one or more A of one or more other monomers; or 4) combinations of 1) to 3) in protein complexes comprising or consisting of two or more monomers. x of Px is for example at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten monomers, or 2 to 60, 3 to 55, 4 to 50, 5 to 45, 6 to 40, 7 to 35, 8 to 30, 9 to 25 or 10 to 20 monomers; preferably x is 2, 3 or 4. Examples of crosslinkers (Q(LB)x) comprising three moieties (B) (i.e., x of the crosslinker is 3) reacting with protein complexes comprising a different number of monomers (Px, e.g., P1, P2, P3, P4 etc.) are shown in Fig. 1 and Fig.3A to 3C. Such a crosslinker is for example Q(LB)3. According to the present invention the protein complex comprises or consists of monomers (P) of a number (x), wherein the moieties B of the crosslinker react with one or more monomer(s) as shown in Fig. 1 and Fig. 3A to 3C. The stabilized protein complex of the present invention is for example multi-crosslinked. The monomers of the protein complex form for example one or more linkages F with one or more crosslinker(s). The different types of crosslinker-monomer(s)-linkages F as shown in Fig. 1 and Fig. 3A to 3C are for example combined in a monomer and / or protein complex. According to the present invention monomers are crosslinked, and / or combinations of monomers are crosslinked in a protein complex. The crosslinking leads to the stabilization of the monomer and / or the protein complex. The reactive group (A) of the monomer such as a sulfhydryl group of a cysteine residue is either part of the native amino acid sequence of the monomer or artificially incorporated (natural or non-natural amino acids). Alternatively, the reactive group (A) is a bioorthogonal group artificially incorporated into the monomer. Alternatively, the monomer comprises a combination of a cysteine residue (native and / or artificial) and bioorthogonal group. The crosslinker either directly reacts with the monomer (one component); or a sulfhydryl-reactive group of a monomer is first modified with a bioorthogonal group (two components) and then reacted with the crosslinker. When reactive groups A of the protein monomer(s) forming the protein complex of the present invention are for example cysteine sulfhydryl groups, these are for example modified before the reaction of the crosslinker (two-components). Such modification is for example a chemical reaction of the sulfhydryl group of a cysteine residue (-SH) with a connector (A´-L-B) as shown in Fig. 2, where A´is a bioorthogonal group and B is a sulfhydryl-reactive moiety. After the reaction, the connector forms a reactive group (A), which then contains a biorthogonal group (e.g., alkyne) capable of reacting with a crosslinker containing corresponding biorthogonal groups (B, e.g., azide). The moieties (B) of the crosslinker (Q(LB)x) for example of Q(LB)3 comprise correspondingly a reactive bioorthogonal group (B) to react with the bioorthogonal groups (A) of the protein complex. When the crosslinker (Q(LB)x) reacts with the reactive group (A) via moiety B linkage F is formed as shown in Fig. 2. Alternatively, the crosslinker reacts directly with reactive group A, as shown in Fig 2 as one-component. For example, the crosslinker reacts with an unmodified cysteine sulfhydryl group (-SH), if the method for the production of the protein complex of the present invention does not comprise the optional step of adding for example a bioorthogonal group to -SH. The connector partially corresponds to the crosslinker. It comprises or consists of the reactive moiety (B), a biorthogonal group (A´) and optionally a linker (L). A connector is for example 2-iodo-N-(prop-2-yn-1-yl)acetamide), 2-iodo-N-(pent-4-yn-1-yl)acetamide), or a combination thereof. A reactive group A of the monomer is a biorthogonal group or a sulfhydryl group. A monomer has one reactive group A or more reactive groups A. The reactive groups are identical or different. A moiety B of the crosslinker is a biorthogonal group or a sulfhydryl-reactive group. A crosslinker has at least three moieties B which are identical or different. Bioorthogonal groups are for example selected from the group consisting of azides, alkynes, including dibenzoazacyclooctynes (DBCO), bicyclo[6.1.0]non-4-yne (BCN), triarylphosphines, cyclooctynes, cyclooctenes, tetrazines, cyclopropenes, cyclobutenes, norbornenes and combinations thereof. Examples of bioorthogonal groups are shown in the following: , or a combination thereof, wherein R1is independently selected from H, F, -O(CH2)(CO)OH, -O(CH2)-pBzOH, R2is independently selected from H, -CH3, -(C6H6)CH2COOH, -pyridyl, R3is independently selected from H, -CH3. The biorthogonal group is a reactive group A of the monomer and / or a moiety B of the crosslinker. Examples of sulfhydryl-reactive groups are shown in the following: combination thereof, wherein Z is selected from F, Cl, Br, I, Tos (O−SO2−C6H4−CH3), and Mes (O−SO2−CH3), R is selected from alkyl. When the reactive group A of the monomer and the moiety B of the crosslinker react linkage F is formed. In linkage F, group A and moiety B form a covalent bond. Linkage F is, wherein R is an alkyl, R1is independently selected from H, F, -O(CH2)(CO)OH, -O(CH2)-pBzOH, R2is independently selected from H, -CH3, -(C6H6)CH2COOH, -pyridyl, and R3is independently selected from H, -CH3. The crosslinker reacts with the same monomer or different monomers of a protein complex. A protein complex comprises one or more crosslinker(s): One embodiment of the present invention refers for example to a protein complex of at least two monomers, at least three monomers or at least four monomers wherein all of the at least three moieties (B) of a crosslinker are covalently linked to three or more reactive groups (A) of one monomer. The number of monomers and the number of crosslinkers in the protein complex is not limited (for examples see Fig 3A). The protein complex comprises for example at least one crosslinker having a core structure Q bound via an optional linker component (L) to at least three moieties (B) which are for example sulfhydryl-reactive groups (e.g., iodoacetyl and / or chloroacetyl) and / or bioorthogonal groups (e.g., azide). The moieties (B) of the crosslinker (Q(LB)x), preferably Q(LB)3, react with corresponding reactive groups (A) for example a sulfhydryl-reactive group (-SH) of a cysteine residue and / or a bioorthogonal group (e.g., alkyne), optionally introduced via chemical modification of a cysteine residue or incorporation of a non-natural amino acid. Reactive group (A) of the protein complex and moiety (B) of the crosslinker form linkage F, e.g., in the final stabilized protein complex. The protein complex comprises or consists of for example two monomers (P1, P2), three monomers (P1, P2, P3), or four monomers (P1, P2, P3, P4). The monomers in these complexes can be the same or different. A second embodiment of the present invention relates to a protein complex of at least two monomers, at least three monomers or at least four monomers wherein one or more of the at least three moieties (B) of the crosslinker are covalently linked to one or more reactive groups (A) of one monomer and one or more of the at least three moieties (B) are covalently linked to one or more reactive groups (A) of one or more other monomers. The number of monomers and the number of crosslinkers is not limited (for examples see Fig 3B). The protein complex comprises for example at least one crosslinker having a core structure Q bound via an optional linker component (L) to at least three moieties (B) which are for example sulfhydryl-reactive groups (e.g., iodoacetyl and / or chloroacetyl) and / or bioorthogonal groups (e.g., azide). The moieties (B) of the crosslinker (Q(LB)x), preferably Q(LB)3, react with corresponding reactive groups (A) for example a sulfhydryl group (-SH) of a cysteine residue and / or a bioorthogonal group (e.g., alkyne), optionally introduced via chemical modification of a cysteine residue or incorporation of a non- natural amino acid. Reactive group (A) of the protein complex and moiety (B) of the crosslinker form linkages F, e.g., in the final stabilized protein complex. The protein complex comprises or consist of for example two monomers (P1, P2), three monomers (P1, P2, P3), or four monomers (P1, P2, P3, P4). The monomers in these complexes can be the same or different. A third embodiment of the present invention relates to a protein complex of at least three monomers or at least four monomers, wherein each of the at least three moieties (B) of the crosslinker is covalently linked to a reactive group (A) of a different monomer. The number of monomers and the number of crosslinkers is not limited (for examples see Fig 3C). The protein complex comprises for example at least one crosslinker having a core structure Q bound via an optional linker component (L) to at least three moieties (B) which are for example sulfhydryl-reactive groups (e.g., iodoacetyl and / or chloroacetyl) and / or bioorthogonal groups (e.g., azide). The moieties (B) of the crosslinker (Q(LB)x), preferably Q(LB)3, react with corresponding reactive groups (A) for example a sulfhydryl group (-SH) of a cysteine residue and / or a bioorthogonal group (e.g., alkyne), optionally introduced via chemical modification of a cysteine residue or incorporation of a non- natural amino acid. Reactive group (A) of the protein complex and moiety (B) of the crosslinker form linkages F, e.g., in the final stabilized protein complex. The protein complex comprises or consist of for example three monomers (P1, P2, P3), or four monomers (P1, P2, P3, P4). The monomers in these complexes can be the same or different. A fourth embodiment of the present invention relates to a protein complex of at least two monomers, at least three monomers or at least four monomers comprising at least two crosslinkers, wherein the at least three moieties (B) of each crosslinker are covalently linked to a reactive group (A) according to embodiment 1, embodiment 2, embodiment 3 or a combination thereof. A crosslinker of the present invention comprises a core Q bound to at least three reactive moieties (B) of the crosslinker wherein Q is either directly bound to B or optionally via a linker component (L). Q is for example selected from the group consisting of or a combination thereof, wherein n is an integer of 1, 2, 3, 4 or 5. Each dashed line in Q indicates a site where Q is optionally bound to L or directly to B. Alternatively Q is for example selected from the group consisting of wherein n is an integer of 1, 2, 3, 4 or 5. Each dashed line in Q indicates a site where Q is optionally bound to L or directly to B. L is for example selected from the group consisting of , , , , or a combinaton thereof wherein X is selected from NH or O, Y is selected from NH or O, Z is selected from NH or O, m is an integer of 1, 2, 3, 4 or 5, p is an integer of 1, 2, 3, 4 or 5, o is an interger of 1, 2, 3 or 4. Each dashed line in L indicates a site where L is bound to Q or B. B is for example selected from the group consisting of or a combination thereof wherein Z is selected from F, Cl, Br, I, Tos (O−SO2−C6H4−CH3), and Mes (O−SO2−CH3), and / or wherein R1is independently selected from H, F, -O(CH2)(CO)OH, -O(CH2)-pBzOH, R2is independently selected from H, -CH3, -(C6H6)CH2COOH, -pyridyl, R3is independently selected from H, -CH3. The biorthogonal group is a reactive group A of the monomer and / or a moiety B of the crosslinker. Each dashed line in B indicates a site where B is optionally bound to L or directly to Q. Alternatively B is , wherein Z is selected from Cl or I. In a protein complex of the present invention Q is for example selected from the group consisting of or a combination thereof, wherein n is an integer of 1, 2, 3, 4 or 5, wherein a dashed line indicates a site where Q is optionally bound to L or directly bound to B; L is optionally selected from the group consisting of or a combinaton thereof wherein X is selected from NH or O, Y is selected from NH or O, Z is selected from NH or O, m is an integer of 1, 2, 3, 4 or 5, p is an integer of 1, 2, 3, 4 or 5, o is an interger of 1, 2, 3 or 4, each dashed line in L indicates a site where L is bound to Q or B; and B is selected from the group consisting of , or a combination thereof, wherein R1is selected from H, F, -O(CH2)(CO)OH, -O(CH2)-pBzOH, R2is selected from H, -CH3, -(C6H6)CH2COOH, -pyridyl, R3is selected from H, -CH3. In another protein complex of the present invention Q is selected from the group consisting of or a combination thereof, wherein n is an integer of 1, 2, 3, 4 or 5, wherein the dashed line indicates a site where Q is optionally bound to L or directly to B; L is optionally selected from the group consisting of wherein X is selected from NH or O, Y is selected from NH or O, Z is selected from NH or O, m is an integer in the range of 1, 2, 3, 4 or 5, p is an integer in the range of 1, 2, 3, 4 or 5, o is an interger in the range of 1, 2, 3 or 4, wherein the dashed line indicates a site where L is bound to Q or B; and B is , wherein Z is F, Cl, Br or I, preferably I; each dashed line in B indicates a site where B is optionally bound to L or directly to Q. In another protein complex of the present invention Q is selected from the group consisting of combination thereof wherein n is an integer of 1, 2, 3, 4 or 5, wherein the dashed line indicates a site where Q is optionally bound to L or directly to B; L is optionally selected from the group consisting of , , , , wherein X is selected from NH or O, Y is selected from NH or O, Z is selected from NH or O, m is an integer in the range of 1, 2, 3, 4 or 5, p is an integer in the range of 1, 2, 3, 4 or 5, o is an interger in the range of 1, 2, 3 or 4, wherein the dashed line indicates a site where L is bound to Q or B; and B is , wherein Z is Cl or I; each dashed line in B indicates a site where B is optionally bound to L or directly to Q. A monomer of the protein complex has for example 50 to 100 amino acids, 60 to 90 amino acids, 70 to 80 amino acids, in particular at least 70 amino acids, at least 80 amino acids, at least 90 amino acids or even at least 100 amino acids. The protein complex and the monomer, respectively, is for example folded or not denatured before cross-linking. As explained herein, the main object of the present invention is to provide a protein complex where a native fold and / or structure is crosslinked, i.e., stabilized. In the protein complex of the present invention the stability of the (natural) tertiary and / or quaternary structure of the monomer(s) is increased compared to the non-cross-linked monomer(s). Consequently, the stability of the (natural) tertiary and / or quaternary structure of the protein complex and monomer, respectively, is increased compared to the non-crosslinked protein complex. Protein structure prediction techniques are well-known in the art and include homology modeling and threading, as well as more advanced methods that utilize neural networks, hidden Markov models and support vector machines. In addition, the tertiary and quaternary structures of a protein can be determined by known methods such as cryoEM X-ray crystallography or nuclear magnetic resonance (NMR) studies. Publicly available software such as the Rosetta software and AlphaFold can also be used for proteins structure prediction and to design new structures. The cysteine residues or biorthogonal groups for crosslinking are for example located in at least two distinct, secondary structures of one or more monomer(s); alternatively, the residues or biorthogonal groups for crosslinking are located in the same secondary structure of two or more monomer(s). For example, the first cysteine residue or biorthogonal group may be located in a first alpha helix and the second residue or biorthogonal group may be located in a second alpha-helix of one or more monomer(s). The third residue or biorthogonal group may be located in either the first or second alpha helix or in a further secondary structure of one or more monomer(s). Such methods have the advantage that the crosslinking increases the stability of at least two secondary structures of one or more monomer(s). More preferably, the three residues or biorthogonal groups for crosslinking are located in at least three distinct secondary structures of one or more monomer(s). The three residues or biorthogonal groups are suitably located within the protein complex so that the crosslinker disclosed herein forms covalent bonds with each of the three cysteine residues or biorthogonal groups. The cysteine residues or biorthogonal groups are for example separated in the primary amino acid sequence by at least 3 amino acids, while still being in spatial proximity in the protein complex. The three cysteine residues or biorthogonal groups form for example a triangle with side lengths between 6 to 38 Angstrom in the protein complex. The cysteine residues or biorthogonal groups are facing for example the same side or different sides of the monomer(s) and of the protein complex, respectively. Positions for the cysteine residues or bioorthogonal groups of the monomer(s) and the protein complex, respectively, are not buried or core positions. "Buried position” as used herein refers to positions that are in the interior of a protein complex or monomer and / or which are inaccessible or nearly inaccessible to solvent. The accessible surface area of a protein complex or monomer is for example determined by a number of different prediction methods. The cysteine residues or bioorthogonal groups are for example located on the surface of the monomer(s) or the protein complex. As used herein, “tertiary structure” of a protein complex or monomer refers to the three- dimensional shape of a protein complex or monomer. The “quaternary structure” of a protein complex or monomer refers to the association of several protein chains or monomers, respectively, into a closely packed arrangement. Each of the monomers has its own primary, secondary, tertiary and quaternary structure. The multivalent sulfhydryl-reactive crosslinker such as a trivalent sulfhydryl-reactive crosslinker comprises for example a fluorophore or an affinity handle. Suitable fluorophores are well-known in the art and include Alexa Fluor 350, Alexa Fluor 405, AMCA, Marina Blue dye, and Cascade Blue dye (available, e.g., from Invitrogen). Affinity handles refers to molecules that can be used for detection and / or purification. Suitable affinity handles are known in the art and may include an antibody, a double- stranded DNA sequence, modified nucleic acids and nucleic acid mimics such as peptide nucleic acids, locked nucleic acids, phosphorodiamidate morpholino oligomers (PMO), a ligand, a receptor, a peptide, or a small molecule for which a cognate binding agent is readily available. Suitable affinity tags are peptide “tags” such as polyhistidine, Calmodulin, S-tag, SBP-tag, Strep-tag, V5, FLAG, HA and Myc tags. Other suitable affinity tags are well-known in the art. The crosslinking reaction is carried out under conditions known in the art. In general, the reaction is carried out at a pH between 5-9 and at a temperature of between 4-60°C. Optionally, the efficiency and / or specificity of the crosslinking reaction can be determined, e.g., using MS or SDS PAGE. The crosslinking reaction is for example carried out under conditions which do not disrupt the tertiary and / or quaternary structure of the protein complex or monomer. The present invention further refers to a composition such as a pharmaceutical composition. The composition such as a pharmaceutical composition comprises for example a protein complex of the present invention and a carrier, excipient, dilutant, such as a pharmaceutically acceptable carrier, excipient, dilutant or stimulant such as an adjuvant, or a combination thereof. In an embodiment the present invention is directed to a method for the production of a protein complex wherein all (e.g., at least three) moieties B of the crosslinker react with at least three A of one monomer comprising for example the steps of a) providing a monomer comprising at least three cysteine sulfhydryl groups or bioorthogonal groups, b) optionally performing a reaction with a connector to add a bioorthogonal group to one or more of the at least three cysteine residues of the monomer isolated or in the protein complex, and c) contacting the monomer or protein complex with a crosslinker comprising a core Q bound to at least three reactive moieties (B) such that each moiety forms a linkage (F) with one of the at least three cysteine residues or bioorthogonal group(s). In a second embodiment, the present invention further refers to a method for the production of a protein complex wherein each moiety B of the crosslinker reacts with a group A of a different monomer comprising the steps of a) providing at least three monomers, for example which are identical or different, each monomer comprising a cysteine sulfhydryl group or a bioorthogonal group, b) optionally performing a reaction with a connector to add a bioorthogonal group to one or more of the at least three cysteine residues of the isolated monomer or in the protein complex, and c) contacting the at least three monomers or the protein complex with a crosslinker comprising a core Q bound to at least three moieties (B) such that each moiety forms a linkage (F) with the cysteine residue or bioorthogonal group of each of the at least three monomers. In a third embodiment, the present invention further refers to a method for the production of a protein complex wherein one or more moieties B of the crosslinker react with one or more group(s) A of one or more other monomers comprising the steps of a) providing at least two monomers in a protein complex, for example which are identical or different, each of the two monomers comprising at least one cysteine sulfhydryl- reactive group or bioorthogonal group, b) optionally performing a reaction with a connector to add a bioorthogonal group to one or more of the at least three cysteine residues of monomers or the protein complex, and c) contacting the monomers or the protein complex with a crosslinker comprising a core Q bound to at least three moieties (B) such that each moiety forms a linkage (F) with the cysteine residue or bioorthogonal group of each of the at least three monomers. In a fourth embodiment, the present invention is directed to the method for the production of a protein complex wherein all (e.g., at least three) moieties B of the crosslinker react with at least three A of only one monomer comprising for example the steps of a) providing a monomer comprising at least three cysteine sulfhydryl-reactive groups or bioorthogonal groups, b) optionally performing a reaction with a connector to add a bioorthogonal group to one or more of the at least three cysteine residues of the monomer isolated or in the protein complex, and c) contacting the monomer or protein complex with a crosslinker comprising a core Q bound to at least three reactive moieties (B) such that each moiety forms linkage (F) with one of the at least three cysteine residues or bioorthogonal group(s); to the method for the production of a protein complex wherein each moiety B of the crosslinker reacts with a group A of a different monomer comprising the steps of a) providing at least three monomers, for example which are identical or different, each monomer comprising a cysteine sulfhydryl-reactive group or a bioorthogonal group, b) optionally performing a reaction with a connector to add a bioorthogonal group to one or more of the at least three cysteine residues of the isolated monomer or in the protein complex, and c) contacting the at least three monomers or the protein complex with a crosslinker comprising a core Q bound to at least three moieties (B) such that each moiety forms a linkage (F) with the cysteine residue or bioorthogonal group of each of the at least three monomers; to the method for the production of a protein complex wherein one or more moieties B of the crosslinker reacts with one or more group(s) A of one or more other monomers comprising the steps of a) providing at least two monomers in a protein complex, for example which are identical or different, each of the two monomers comprising at least one cysteine sulfhydryl- reactive group or bioorthogonal group, b) optionally performing a reaction with a connector to add a bioorthogonal group to one or more of the at least three cysteine residues of monomers or the protein complex, and c) contacting the monomers or the protein complex with a crosslinker comprising a core Q bound to at least three moieties (B) such that each moiety forms a linkage (F) with the cysteine residue or bioorthogonal group of each of the at least three monomers; or a combination thereof. Combinations of the methods result in different combinations of monomers in the protein complex. The method of producing the protein complex comprises modifying the protein(s) in step a) to introduce one or more of the cysteine residues in the monomer or in one or more of the monomers. The two or more monomer(s) of the protein complex comprise for example one or more additional cysteine residue(s), which do not react with the crosslinker, in particular the moieties (B) of the crosslinker. Thus, the method of the present invention does not result in the formation of a covalent bond between the one or more additional cysteine residue(s) and B. The one or more additional cysteine residue(s) are for example protected, e.g., by a ligand such as a small molecule, a cofactor, an ion, a monomer, a protein domain or a combination thereof. This avoids the reaction of the crosslinker with such additional cysteine residue and results in a controlled crosslinking of selected cysteine residues. A protein complex of the present invention and the method of its production, respectively, reduces or even avoids the propensity for aggregation of the protein complex of the present invention (see Fig. 15 to Fig. 17). The increased stability of proteins upon crosslinking according to the present invention is based, e.g., at least partially, on a reduced aggregation. Aggregation is a problem for a number of applications for example it can result in precipitation and thus, inactivation. Aggregation leads for example to reduced activity of enzymes, to impaired functionality of receptors, antigens, or antibodies, and to increased immunogenicity by therapeutic proteins. The stability of the protein complex is for example determined by measuring turbidity by back-reflection or particle size by dynamic light scattering (DLS), giving information about the presence of large and small aggregates, particle size distribution and polydispersity. Typically, the higher the temperature of turbidity onset, the lack of change in turbidity, or a low distribution of size populations, the more stable is the monomer and / or the protein complex. The delayed onset or lack of aggregation prolongs for example enzymatic activity. The delayed onset or lack of aggregation increases for example efficacy and safety of therapeutic proteins. A therapeutic protein is for example an antibody, an antigen, an enzyme, a receptor or a combination thereof. Such a therapeutic protein is for example a vaccine. Protein aggregates have been reported to be associated with adverse immune responses in patients (e.g., immunogenicity), including the development of anti-drug antibodies (ADAs) which in turn adversely affect the pharmacokinetics, bioavailability, and efficacy of therapeutic proteins (see, e.g., MG Tovey, C Lallemand Immunogenicity and other problems associated with the use of biopharmaceuticals Ther. Adv. Drug. Saf., 2011, 2, 113-28; MLE Lundahl, S. Fogli, PE Colavita, EM Scanlan Aggregation of protein therapeutics enhances their immunogenicity: causes and mitigation strategies RSC Chem. Biol., 2021, 2, 1004-1020; EM Moussa, JP Panchal, BS Moorthy, JS Blum, MK Joubert, LO Narhi, EM Topp Immunogenicity of therapeutic protein aggregates J. Pharm. Sci., 105, 2016, 417e430). Embodiments of the present invention are for example 1. Protein complex comprising or consisting of two or more monomers in native tertiary and / or quaternary structure which is fixed by a crosslinker having a core structure Q and at least three moieties B covalently linked to reactive groups A in one or more of the monomers forming a linkage F, wherein group A is independently a sulfhydryl group or a bioorthogonal group, and moiety B is independently a sulfhydryl-reactive group or a bioorthogonal group. 2. Protein complex according to embodiment 1 wherein the protein complex comprises or consists of 2 to 60 monomers. 3. Protein complex according to embodiment 1 or 2 wherein the protein complex is a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, or an octamer. 4. Protein complex according to any one of embodiments 1 to 3 wherein the protein complex is a dimer. 5. Protein complex according to any one of embodiments 1 to 3 wherein the protein complex is a trimer. 6. Protein complex according to any one of embodiments 1 to 3 wherein the protein complex is a tetramer. 7. Protein complex according to any one of embodiments 1 to 6, wherein A is a sulfhydryl group and B is a sulfhydryl-reactive group. 8. Protein complex according to any one of embodiments 1 to 6, wherein A is a bioorthogonal group and B is a bioorthogonal group. 9. Protein complex according to any one of embodiments 1 to 6, wherein A is a sulfhydryl group and B is a bioorthogonal group. 10. Protein complex according to any one of embodiments 1 to 6, wherein A is a bioorthogonal group and B is a sulfhydryl-reactive group. 11. Protein complex according to any one of embodiments 1 to 10 wherein the linkage F comprises or consists of group A covalently bound to moiety B. 12. Protein complex according to any one of embodiments 1 to 11 wherein the linkage F

[0002] wherein R is an alkyl, R1is independently selected from H, F, -O(CH2)(CO)OH, -O(CH2)-pBzOH, R2is independently selected from H, -CH3, -(C6H6)CH2COOH, -pyridyl, and R3is independently selected from H, -CH3. 13. Protein complex according to any one of embodiments 1 to 12 wherein the crosslinker has three or more, or four or more, five or more, six or more, seven of more, eight or more, nine or more, or ten or more moieties B. 14. Protein complex according to any one of embodiments 1 to 13 wherein the crosslinker has three moieties B. 15. Protein complex according to any one of embodiments 1 to 14 wherein each moiety B of the crosslinker is covalently linked to a different reactive group A of a different monomer. 16. Protein complex according to any one of embodiments 1 to 14 wherein all of the at least three moieties (B) of a crosslinker are covalently linked to three or more reactive groups (A) of one monomer. 17. Protein complex according to any one of embodiments 1 to 14 wherein one or more of the at least three moieties (B) of the crosslinker are covalently linked to one or more reactive groups (A) of one monomer, and one or more of the at least three moieties (B) are covalently linked to one or more reactive groups (A) of one or more other monomer(s). 18. Protein complex according to any one of embodiments 1 to 14 wherein each moiety B of the crosslinker is covalently linked to a different reactive group A of a different monomer, wherein all of the at least three moieties (B) of a crosslinker are covalently linked to three or more reactive groups (A) of one monomer, wherein one or more of the at least three moieties (B) of the crosslinker are covalently linked to one or more reactive groups (A) of one monomer, and one or more of the at least three moieties (B) are covalently linked to one or more reactive groups (A) of one or more other monomer(s) or a combination thereof. 19. Protein complex according to any one of embodiments 1 to 18 wherein the protein complex is multi-stabilized comprising two or more monomers covalently linked to two or more crosslinkers. 20. Protein complex according to any one of embodiments 1 to 19 comprising or consisting of at least two monomers wherein all of the at least three moieties (B) of a crosslinker are covalently linked to three or more reactive groups (A) of one monomer. 21. Protein complex according to any one of embodiments 1 to 19 comprising or consisting of at least two monomers wherein one or more of the at least three moieties (B) of the crosslinker are covalently linked to one or more reactive groups (A) of one monomer and one or more of the at least three moieties (B) are covalently linked to one or more reactive groups (A) of one or more other monomer(s). 22. Protein complex according to any one of embodiments 1 to 19 comprising or consisting of at least three monomers wherein each of the at least three moieties (B) of the crosslinker is covalently linked to a reactive group (A) of different monomers. 23. Protein complex according to any one of embodiments 1 to 19 comprising or consisting of at least two monomers wherein all of the at least three moieties (B) of a crosslinker are covalently linked to three or more reactive groups (A) of one monomer, comprising or consisting of at least two monomers wherein one or more of the at least three moieties (B) of the crosslinker are covalently linked to one or more reactive groups (A) of one monomer and one or more of the at least three moieties (B) are covalently linked to one or more reactive groups (A) of one or more other monomer(s), comprising or consisting of at least three monomers wherein each of the at least three moieties (B) of the crosslinker is covalently linked to a reactive group (A) of different monomers or a combination thereof. 24. Protein complex according to any one of embodiments 20, 21 or 23 wherein the protein complex comprises or consists of at least three monomers. 25. Protein complex according to any one of embodiments 20 to 23 wherein the protein complex comprises or consists of at least four monomers. 26. Protein complex according to any one of embodiments 20 to 24 wherein the protein complex comprises or consists of three monomers 26. Protein complex according to any one of embodiments 20 to 25 wherein the protein complex comprises or consists of four monomers. 27. Protein complex according to any one of embodiments 1 to 26 fixed via the crosslinker wherein the core structure Q is directly bound to B or optionally via linker component L. 28. Protein complex according to any one of embodiments 1 to 27 wherein Q is selected from the group consisting of , or a combination thereof, wherein n is an integer of 1, 2, 3, 4 or 5, wherein a dashed line indicates a site where Q is directly bound to B or optionally via L. 29. Protein complex according to any one of embodiments 1 to 28 wherein Q is selected from the group consisting of wherein n is an integer of 1, 2, 3, 4 or 5; each dashed line in Q indicates a site where Q is optionally bound to L or directly to B. 30. Protein complex according to any one of embodiments 1 to 29 wherein optional L is selected from the group consisting of wherein X is selected from NH or O, Y is selected from NH or O, Z is selected from NH or O, m is an integer of 1, 2, 3, 4 or 5, p is an integer of 1, 2, 3, 4 or 5, o is an interger in the range of 1, 2, 3 or 4, wherein the dashed line indicates a site where L is bound to Q or B. 31. Protein complex according to any one of embodiments 1 to 30 wherein B is selected from the group consisting of wherein Z is selected from F, Cl, Br, I, Tos (O−SO2−C6H4−CH3), and Mes (O−SO2−CH3), R1is selected from H, F, -O(CH2)(CO)OH, -O(CH2)-pBzOH, R2is selected from H, -CH3, -(C6H6)CH2COOH, -pyridyl, R3is selected from H, -CH3, wherein each dashed indicates a site where B is bound to the linker L. 32. Protein complex according to embodiment 31 wherein B is selected from the group consisting of or a combination thereof wherein Z is selected from F, Cl, Br, I, Tos (O−SO2−C6H4−CH3), and Mes (O−SO2−CH3). 33. Protein complex according to embodiment 31 or 32 wherein B is , wherein Z is selected from F, Cl, Br, I, Tos (O−SO2−C6H4−CH3), and Mes (O−SO2−CH3). 34. Protein complex according to any one of embodiments 31 to 33 wherein Z is Cl or I. 35. Protein complex according to embodiment 31 wherein B is selected from the group consisting of wherein R1is independently selected from H, F, -O(CH2)(CO)OH, -O(CH2)-pBzOH, R2is independently selected from H, -CH3, -(C6H6)CH2COOH, -pyridyl, R3is independently selected from H, -CH3. The biorthogonal group is a reactive group A of the monomer and / or a moiety B of the crosslinker; each dashed line in B indicates a site where B is optionally bound to L or directly to Q. 36. Protein complex according to any one of embodiments 1 to 35 wherein Q is selected from the group consisting of or a combination therof, wherein n is an integer of 1, 2, 3, 4 or 5, wherein each dashed line in Q indicats a site where Q is directly bound to B or optionally via L; L is optionally selected from the group consisting of , or a combination thereofwherein X is selected from NH or O, Y is selected from NH or O, Z is selectedfrom NH or O, m is an integer of 1, 2, 3, 4 or 5, p is an integer of 1, 2, 3, 4 or 5, o is an interger in the range of 1, 2, 3 or 4, wherein the dashed line indicates a site where L is bound to Q or B; and B is selected from the group consisting of combination thereof, wherein Z is selected from F, Cl, Br, I, Tos (O−SO2−C6H4−CH3), and Mes (O−SO2−CH3), wherein each dashed line in B indicates a site where B is optionally bound to L or directly to Q. 37. Protein complex according to any one of embodiments 1 to 35 wherein Q is selected from the group consisting of , or a combination thereof, wherein n is an integer of 1, 2, 3, 4 or 5, wherein each dashed line in Q is optionally bound to L or directly to B; L is optionally selected from the group consisting of , , , , , or a combination thereofwherein X is selected from NH or O, Y is selected from NH or O, Z is selectedfrom NH or O, m is an integer of 1, 2, 3, 4 or 5, p is an integer in the range of 1, 2, 3, 4 or 5, o is an interger in the range of 1, 2, 3 or 4, wherein each dashed line indicates a site where L is bound to Q or B, and B is selected from the group consisting of , R1is selected from H, F, -O(CH2)(CO)OH, -O(CH2)-pBzOH, R2is selected from H, -CH3, -(C6H6)CH2COOH, -pyridyl, R3is selected from H, -CH3, wherein each dashed line in B indicates a site where B is optionally bound to L or directly to Q. 38. Protein complex according to any one of embodiments 1 to 35 wherein Q is selected from the group consisting of , or a combination thereof, wherein n is an integer of 1, 2, 3, 4 or 5, wherein each dashed line in Q indicates a site where Q is optionally bound to L or directly bound to B; L is optionally selected from the group consisting of or a combination thereof wherein X is selected from NH or O, Y is selected from NH or O, Z is selectedfrom NH or O, m is an integer of 1, 2, 3, 4 or 5, p is an integer of 1, 2, 3, 4 or 5, o is an interger of 1, 2, 3, 4 or 5, wherein the dashed line indicates a site where L is bound to Q or B; and B is , wherein Z is F, Cl, Br or I, preferably I, wherein the dashed line in B indicates a site where B is optionally bound to L of directly bound to Q. 39. Protein complex according to embodiment 38, wherein B is wherein Z is Cl. 40. Protein complex according to embodiment 38, wherein B is , wherein Z is I. 41. Protein complex according to any one of embodiments 1 to 40 wherein the protein complex is an enzyme, a cytokine, a chemokine, a growth factor, a hormone, an antibody, a receptor, an antigen, structural protein, a defensive protein, a storage protein, a transport protein, a contractile, a signaling, a membrane, a secreted protein or combinations thereof. 42. Protein complex according to embodiment 41 wherein the protein complex is an enzyme. 43. Protein complex according to embodiment 41 wherein the protein complex is a receptor. 44. Protein complex according to any one of embodiments 41 to 43 which is a therapeutic compound. 45. Protein complex according to any one of embodiments 1 to 44 for use in a medical treatment. 46. Protein complex according to any one of embodiments 1 to 45 having increased thermal and / or chemical stability. 47. Protein complex according to any one of embodiments 1 to 46 wherein the bioorthogonal group is selected from the group consisting of azides, alkynes, including dibenzoazacyclooctynes (DBCO), bicyclo[6.1.0]non-4-yne (BCN), triarylphosphines, cyclooctynes, cyclooctenes, tetrazines, cyclopropenes, cyclobutenes, norbornenes and combinations thereof. 48. Protein complex according to any one of embodiments 1 to 47 wherein the sulfhydryl- reactive moiety B is iodoacetyl and / or chloroactyl. 49. Method for preparing a protein complex according to any one of embodiments 1 to 47 comprising or consisting of the steps: a) providing a protein complex in its tertiary and / or quaternary structure, wherein two or more of the monomers of the protein complex comprise at least three reactive groups A, b) adding a crosslinker comprising three or more moieties B which form a linkage F with the reactive groups A of the monomers, wherein A is independently a sulfhydryl group or a bioorthogonal group and B is independently a sulfhydryl-reactive moiety or a bioorthogonal moiety, and the crosslinker increases the stability of the native tertiary and / or quaternary structure of the protein complex and / or monomer. 50. Method according to embodiment 49 comprising or consisting of the steps: a) providing a protein complex in its tertiary and / or quaternary structure, wherein one monomer comprises at least three cysteine sulfhydryl groups A and / or bioorthogonal groups A, b) optionally performing a reaction with a connector to add a bioorthogonal group to one or more of the at least three cysteine residues of the monomer, and c) contacting the monomer with a crosslinker comprising a core Q bound to at least three reactive moieties (B) such that each moiety forms a linkage (F) with one of the at least three cysteine residues or bioorthogonal group(s) of the monomer. 51. Method according to embodiment 49 comprising or consisting of the steps: a) providing a protein complex in its tertiary and / or quaternary structure, wherein at least three monomers comprise a cysteine sulfhydryl group A and / or a bioorthogonal group A, b) optionally performing a reaction with a connector to add a bioorthogonal group to one or more of the at least three cysteine residues of the monomer and c) contacting the at least three monomers with a crosslinker comprising a core Q bound to at least three moieties (B) such that each moiety forms a linkage (F) with the cysteine residue or bioorthogonal group of each of the at least three monomers. 52. Method according to embodiment 49 comprising or consisting of the steps: a) providing a protein complex in its tertiary and / or quaternary structure, wherein at least two monomers, each comprise at least one cysteine sulfhydryl group A and / or bioorthogonal group A, b) optionally performing a reaction with a connector to add a bioorthogonal group to one or more of the at least three cysteine residues of the monomers, and c) contacting the at least two monomers with a crosslinker comprising a core Q bound to at least three moieties (B) such that each moiety forms a linkage (F) with the cysteine residue or bioorthogonal group of each of the at least two monomers. 53. Method according to embodiment 49 comprising or consisting of the steps a) providing a protein complex in its tertiary and / or quaternary structure, wherein one monomer comprises at least three cysteine sulfhydryl groups A and / or bioorthogonal groups A, b) optionally performing a reaction with a connector to add a bioorthogonal group to one or more of the at least three cysteine residues of the monomer, and c) contacting the monomer with a crosslinker comprising a core Q bound to at least three reactive moieties (B) such that each moiety forms a linkage (F) with one of the at least three cysteine residues or bioorthogonal group(s) of the monomer; a) providing a protein complex in its tertiary and / or quaternary structure, wherein at least three monomers comprise a cysteine sulfhydryl group A and / or a bioorthogonal group A, b) optionally performing a reaction with a connector to add a bioorthogonal group to one or more of the at least three cysteine residues of the monomer and c) contacting the at least three monomers with a crosslinker comprising a core Q bound to at least three moieties (B) such that each moiety forms a linkage (F) with the cysteine residue or bioorthogonal group of each of the at least three monomers; a) providing a protein complex in its tertiary and / or quaternary structure, wherein at least two monomers, each comprise at least one cysteine sulfhydryl group A and / or bioorthogonal group A, b) optionally performing a reaction with a connector to add a bioorthogonal group to one or more of the at least three cysteine residues of the monomers, and c) contacting the at least two monomers with a crosslinker comprising a core Q bound to at least three moieties (B) such that each moiety forms a linkage (F) with the cysteine residue or bioorthogonal group of each of the at least two monomers; or a combination thereof. 54. Method according to any one of embodiments 49 to 53 wherein the connector comprises or consists of a bioorthogonal group A´, a component L and a moiety B reacting with the reactive group A of the monomer. 55. Method according to any one of embodiments 49 to 54 wherein the connector is 2- iodo-N-(prop-2-yn-1-yl)acetamide), 2-iodo-N-(pent-4-yn-1-yl)acetamide), or a combination thereof. 56. Method according to any one of embodiments 49 to 55 wherein the reactive sulfhydryl-reactive group and / or bioorthogonal group A is introduced in the monomer via chemical modification of a cysteine residue or incorporation of a non-natural amino acid. 57. Protein complex according to any one of embodiments 1 to 48 or method according to any one of embodiments 49 to 56 wherein the bioorthogonal group is selected from the group consisting of wherein R1is selected from H, F, -O(CH2)(CO)OH, -O(CH2)-pBzOH R2is selected from H, -CH3, -(C6H6)CH2COOH, -pyridyl R3is selected from H, -CH3. 58. Protein complex according to any one of embodiments 1 to 48 or 57, or method according to any one of embodiments 49 to 57 wherein the bioorthogonal group is selected from the group consisting of azides, alkynes, including dibenzoazacyclooctynes (DBCO), bicyclo[6.1.0]non-4-yne (BCN), triarylphosphines, cyclooctynes, cyclooctenes, tetrazines, cyclopropenes, cyclobutenes, norbornenes and combinations thereof. 59. Method according to any one of embodiments 49 to 58 wherein the sulfhydryl- reactive group is iodoacetyl and / or chloroacetyl. 60. Method according to any one of embodiments 49 to 59 wherein a sulfhydryl group A is introduced in the monomer which reacts with an electrophilic group of the connector comprising the bioorthogonal group to introduce the bioorthogonal group A in the monomer. 61. Method according to any one of embodiments 49 to 60 wherein the sulfhydryl- reactive moiety B of the crosslinker forms a covalent linkage with the sulfhydryl group A of the monomer. 62. Method according to any one of embodiments 49 to 60 wherein the bioorthogonal moiety B of the crosslinker forms a covalent linkage with the bioorthogonal group A of the monomer. 63. Method according to any one of embodiments 49 to 59 wherein the sulfhydryl- reactive moiety B of the crosslinker forms a covalent linkage with the sulfhydryl group A of the monomer, wherein the bioorthogonal moiety B of the crosslinker forms a covalent linkage with the bioorthogonal group A of the monomer, or a combination thereof in one or more monomers of the protein complex. 64. Protein complex according to any one of embodiments 1 to 48, 57 or 58, or method according to any one of embodiments 49 to 63 wherein the crosslinker is selected from the group consisting of 1,1',1''- trioxa-4,10,16-triazacyclooctadecane-4,10,16- triyl)tris(2-chloroethan-1-one), 4,10,16-tris(vinylsulfonyl)-1,7,13-trioxa-4,10,16- triazacyclooctadecane), 1,1',1''-(1,4,7-triazonane-1,4,7-triyl)tris(2-chloroethan-1-one), 1,4,7-tris(vinylsulfonyl)-1,4,7-triazonane), N,N',N''-(benzene-1,3,5- triyltris(methylene))tris(2-chloroacetamide), N,N',N''-(benzene-1,3,5- triyltris(methylene))tris(2-iodoacetamide), N,N′,N′′-((1,3,5-triazinane-1,3,5-triyl)tris(2- oxoethane-2,1-diyl))tris(2-chloroacetamide), N,N',N''-((1,3,5-triazinane-1,3,5-triyl)tris(2- oxoethane-2,1-diyl))tris(2-iodoacetamide), N,N',N''-((1,3,5-triazinane-1,3,5-triyl)tris(2- oxoethane-2,1-diyl))tris(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide), N,N',N''- (benzene-1,3,5-triyltris(methylene))tris(3-(2-chloroacetamido)propanamide), N,N',N''- (benzene-1,3,5-triyltris(methylene))tris(3-(2-iodoacetamido)propanamide), N,N',N''- (benzene-1,3,5-triyltris(methylene))tris(3-(2-(2-chloroacetamido)ethoxy)propanamide), N,N',N''-(benzene-1,3,5-triyltris(methylene))tris(3-(2-(2- iodoacetamido)ethoxy)propanamide), N,N',N''-((1,3,5-triazinane-1,3,5-triyl)tris(2- oxoethane-2,1-diyl))tris(3-(2-chloroacetamido)propanamide), N,N',N''-((1,3,5-triazinane- 1,3,5-triyl)tris(2-oxoethane-2,1-diyl))tris(3-(2-iodoacetamido)propanamide), N,N',N''- ((1,3,5-triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))tris(3-(2-(2- chloroacetamido)ethoxy)propanamide), N,N',N''-((1,3,5-triazinane-1,3,5-triyl)tris(2- oxoethane-2,1-diyl))tris(3-(2-(2-iodoacetamido)ethoxy)-propanamide), N1,N1',N1''-((1,3,5- triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))tris(N6,N6-dicyclobenzoazacyclo- octynylamide), N1,N1',N1''-(nitrilotris(propane-3,1-diyl))tris(N6,N6- dicyclobenzoazacyclooctynylamide), and a combination thereof. 65. Protein complex or method according to embodiment 64 wherein the crosslinker is selected from the group consisting of N,N′,N′′-((1,3,5-triazinane-1,3,5-triyl)tris(2- oxoethane-2,1-diyl))tris(2-chloroacetamide), N,N',N''-((1,3,5-triazinane-1,3,5-triyl)tris(2- oxoethane-2,1-diyl))tris(2-iodoacetamide), N,N',N''-((1,3,5-triazinane-1,3,5-triyl)tris(2- oxoethane-2,1-diyl))tris(3-(2-(2-chloroacetamido)ethoxy)propanamide)), N,N',N''-((1,3,5- triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))tris(3-(2-(2- iodoacetamido)ethoxy)propanamide)), N,N',N''-(benzene-1,3,5-triyltris(methylene))tris(3- (2-(2-chloroacetamido)ethoxy)propanamide), N,N′,N′′-(benzene-1,3,5- triyltris(methylene))tris(2-chloroacetamide), N,N',N''-(benzene-1,3,5- triyltris(methylene))tris(2-iodoacetamide), N,N',N''-(benzene-1,3,5- triyltris(methylene))tris(3-(2-(2-chloroacetamido)ethoxy)propanamide), N,N',N''- (benzene-1,3,5-triyltris(methylene))tris(3-(2-(2-iodoacetamido)ethoxy)propanamide), or a combination thereof. 66. Method according to any one of embodiments 49 to 65 wherein the protein complex is an enzyme, a cytokine, a chemokine, a growth factor, a hormone, an antibody, a receptor, an antigen, structural protein, a defensive protein, a storage protein, a transport protein, a contractile, a signaling, a membrane, a secreted protein or combinations thereof. 67. Method according to embodiment 66 wherein the protein complex is an enzyme. 68. Method according to embodiment 66 wherein the protein complex is a receptor. 69. Method according to any one of embodiments 49 to 68 for crosslinking of protein complex PFE wherein PFE variants are incubated with tris-electrophile Ta4, Ta5, or Bz4. 70. Method according to any one of embodiments 49to 68 for two-component crosslinking protein complex PFE wherein PFE variants are alkylated with connectors IA-C3 (Q3) or IA-C4 (Q5) prior to crosslinking with Ta-N3 (T) or Bz-N3 (B)-azide. 71. Method according to any one of embodiments 49 to 68 for crosslinking of protein complex PFE wherein PFE-T, PFE-Q and PFE-DM variants are incubated with tris- electrophile Ta4, Ta5, or Bz4. 72. Method according to any one of embodiments 49 to 68 for two-component crosslinking of protein complex PFE wherein variant PFE-Q is alkylated with connector IA-C3 (Q3) or IA-C4 (Q5) prior to crosslinking with tris-crosslinkers Ta-N3 (T) or Bz-N3 (B)-azide. 73. Method according to any one of embodiments 69 to 72 wherein the crosslinking buffer is 50 mM HEPES at pH 8.5 and 50 mM NaCl. 74. Crosslinker comprising or consisting of three components: a core (Q), an optional linker (L) and at least three moieties (B). 75. Crosslinker according to embodiment 74 comprising three moieties (B). 76. Crosslinker according to embodiment 74 or 75 selected from the group consisting of trioxa-4,10,16-triazacyclooctadecane-4,10,16-triyl)tris(2-chloroethan-1- one), 4,10,16-tris(vinylsulfonyl)-1,7,13-trioxa-4,10,16-triazacyclooctadecane), 1,1',1''- (1,4,7-triazonane-1,4,7-triyl)tris(2-chloroethan-1-one), 1,4,7-tris(vinylsulfonyl)-1,4,7- triazonane), N,N',N''-(benzene-1,3,5-triyltris(methylene))tris(2-chloroacetamide), N,N',N''-(benzene-1,3,5-triyltris(methylene))tris(2-iodoacetamide), N,N′,N′′-((1,3,5- triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))tris(2-chloroacetamide), N,N',N''-((1,3,5- triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))tris(2-iodoacetamide), N,N',N''-((1,3,5- triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))tris(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)hexanamide), N,N',N''-(benzene-1,3,5-triyltris(methylene))tris(3-(2- chloroacetamido)propanamide), N,N',N''-(benzene-1,3,5-triyltris(methylene))tris(3-(2- iodoacetamido)propanamide), N,N',N''-(benzene-1,3,5-triyltris(methylene))tris(3-(2-(2- chloroacetamido)ethoxy)propanamide), N,N',N''-(benzene-1,3,5- triyltris(methylene))tris(3-(2-(2-iodoacetamido)ethoxy)propanamide), N,N',N''-((1,3,5- triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))tris(3-(2-chloroacetamido)propanamide), N,N',N''-((1,3,5-triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))tris(3-(2- iodoacetamido)propanamide), N,N',N''-((1,3,5-triazinane-1,3,5-triyl)tris(2-oxoethane-2,1- diyl))tris(3-(2-(2-chloroacetamido)ethoxy)propanamide), N,N',N''-((1,3,5-triazinane-1,3,5- triyl)tris(2-oxoethane-2,1-diyl))tris(3-(2-(2-iodoacetamido)ethoxy)-propanamide), N1,N1',N1''-((1,3,5-triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))tris(N6,N6- dicyclobenzoazacyclo-octynylamide), N1,N1',N1''-(nitrilotris(propane-3,1- diyl))tris(N6,N6-dicyclobenzoazacyclooctynylamide), and a combination thereof. 77. Crosslinker according to embodiment 76 wherein the crosslinker is selected from the group consisting of N,N′,N′′-((1,3,5-triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))tris(2- chloroacetamide), N,N',N''-((1,3,5-triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))tris(2- iodoacetamide), N,N',N''-((1,3,5-triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))tris(3-(2- (2-chloroacetamido)ethoxy)propanamide)), N,N',N''-((1,3,5-triazinane-1,3,5-triyl)tris(2- oxoethane-2,1-diyl))tris(3-(2-(2-iodoacetamido)ethoxy)propanamide)), N,N′,N′′-(benzene- 1,3,5-triyltris(methylene))tris(2-chloroacetamide), N,N',N''-(benzene-1,3,5- triyltris(methylene))tris(2-iodoacetamide), N,N',N''-(benzene-1,3,5- triyltris(methylene))tris(3-(2-(2-chloroacetamido)ethoxy)propanamide), N,N',N''- (benzene-1,3,5-triyltris(methylene))tris(3-(2-(2-iodoacetamido)ethoxy)propanamide), N,N',N''-(benzene-1,3,5-triyltris(methylene))tris(2-iodoacetamide), or a combination thereof. 78. Use of a crosslinker in a method according to any one of embodiments 49 to 73. 79. Use of a crosslinker according to embodiment 79 for reacting with a sulfhydryl group A or a bioorthogonal group A of a monomer, preferably for crosslinking three cysteine residues present in the monomer. 80. Protein complex according to any one of embodiments 1 to 48, 57 or 58 wherein Q is selected from the group consisting of combination therof, wherein n is an integer of 1, 2, 3, 4 or 5, wherein each dashed line in Q indicats a site where Q is directly bound to B or optionally via L; L is optionally selected from the group consisting of , or a combination thereofwherein X is selected from NH or O, Y is selected from NH or O, Z is selectedfrom NH or O, m is an integer of 1, 2, 3, 4 or 5, p is an integer of 1, 2, 3, 4 or 5, o is an interger in the range of 1, 2, 3 or 4, wherein the dashed line indicates a site where L is bound to Q or B; and B is selected from the group consisting of combination thereof, wherein Z is selected from F, Cl, Br, I, Tos (O−SO2−C6H4−CH3), and Mes (O−SO2−CH3), wherein each dashed line in B indicates a site where B is optionally bound to L or directly to Q. 81. Use of the protein complex according to any one of embodiments 1 to 48, 57, 58 or 80 for reducing or avoiding formation of aggregates in vitro. 82. Protein complex according to any one of embodiments 1 to 48, 57, 58 or 80 for use in preventing and / or treating formation of aggregates of a therapeutic protein. 83. Protein complex for use according to embodiment 82 wherein the therapeutic protein is an antibody, an antigen, an enzyme, a receptor or a combination thereof. 84. Protein complex for use according to embodiment 82 wherein the therapeutic protein is an antibody. 85. Protein complex for use according to embodiment 82 wherein the therapeutic protein is an antigen. 86. Protein complex for use according to embodiment 82 wherein the therapeutic protein is an enzyme. 87. Protein complex for use according to embodiment 82 wherein the therapeutic protein is a receptor. 88. Protein complex for use according to any one of embodiments 82 to 87 wherein the therapeutic protein is a vaccine. 89. Protein complex for use according to embodiment 88 for preventing an undesired side effect of the vaccine. 90. Protein complex for use according to embodiment 89 wherein the undesired side effect is an immunogenic reaction. 91. Protein complex for use according to embodiment 89 wherein the undesired side effect is pain, swelling of the injection side, redness of the injection side, fever, chill, feeling tired, headache, muscle ach, joint ache, fast heartbeat, difficulty breathing, dizziness, weakness or a combination thereof. 92. Protein complex according to any one of embodiments 1 to 48, 57, 58 or 80 to 91, wherein the protein complex is stabilized by reduced aggregation. 93. Protein complex according to any one of embodiments 1 to 48, 57, 58 or 80 to 92, wherein the protein complex is partially stabilized by reduced aggregation. 94. Protein complex according to any one of embodiments 1 to 48, 57, 58 or 80 to 93 having reduced aggregation. Examples The following examples illustrate different embodiments of the present invention, but the invention is not limited to these examples. General procedures Protein expression In general, chemically competent E. coli (BL21(DE3)) cells are transformed with a pET28 vector containing the genes coding for His6-tagged protein variants. Cells are grown in culture medium, and protein expression is induced with IPTG. After incubation, cells are harvested using centrifugation (Beckman Coulter JLA8.1, 4000 rpm, 20 min, 4 °C) and re- suspended in lysis buffer. Cell lysis is performed using a microfluidizer (Microfluidics LM10) at 15000 psi. The resulting cell lysate is cleared of debris by centrifugation (Beckman Coulter JA25.50, 21000 rpm, 45 min, 4 °C). Protein variant purification generally includes affinity purification, tag cleavage (if desired) and size-exclusion chromatography. Protein containing fractions are pooled, concentrated and flash frozen in liquid nitrogen. Crosslinking / alkylation of cysteine sulfhydryl groups In general, crosslinking is performed with 50–500 µM protein and 0.2–1 mM crosslinker in a pH 7–9 buffer for 1–24 h. Excess crosslinker is removed and buffer exchange performed by column filtration (e.g. spin, desalting, or size exclusion column). Copper-catalyzed azide–alkyne 1,3-dipolar cycloaddition In general, crosslinking is performed with 50–500 µM alkyne (or azide)-bearing protein and 30–1000 µM tris-azide (or tris alkyne) in a pH 6–9 buffer. Premixed CuSO4 (100 μM) with THPTA ligand (500 μM) is added, followed by the addition of sodium ascorbate (5 mM). The reaction is incubated for 1–12 h, excess crosslinker removed and buffer exchange performed by column filtration (e.g., spin, desalting, or size exclusion column). Strain-promoted azide–alkyne cycloaddition In general, crosslinking is performed with 50–500 µM alkyne (or azide)-bearing protein and 0.2–1 mM alkyne (or azide)-bearing crosslinker in a pH 6–9 buffer for 1–24 h. Excess crosslinker is removed and buffer exchange performed by column filtration (e.g. spin, desalting, or size exclusion column). SDS PAGE In general, samples were pre-incubated with 5x SDS loading buffer at 95 °C for 5 min and loaded onto a pre-casted bis-tris 4-20% gel (Genscript). Separation was performed for 1 h at 160 V (Biorad) and staining was done with InstantBlue Coomassie (Abcam). Thermal unfolding In general thermal unfolding is performed using either nanoDSF (Nanotemper Prometheus) or circular dichroism spectrometry (Jasco J-1500) and resulting Tm values (heated from 25–95 °C at rate of 1 °C min–1) and Ti values (Nanotemper Tycho heated from 35–95 °C at rate of 30 °C min–1)according to standard procedures. LC-MS Intact protein mass spectrometry is performed on an Agilent HPLC system connected to either a quadrupole or time-of-flight mass spectromer, using an AdvanceBio RPmAb C4 column and water / acetonitrile with 0.1% formic acid as eluent. Analysis and deconvolution were performed using Openlab CDS Chemstation or MassHunter and Bioconfirm software. Activity assays PFE activity assay: Activity assay buffer (90 µl), protein sample (5 µl of 1 µM) and p- nitrophenyl acetate solution (5 µl of 20 mM in DMSO) were charged in a microtiter plate. Measurement was started immediately over 20 min at RT and a wavelength of 410 nm in 10 s intervals per well (TECAN SPARK 20M plate reader). Blank corrected OD values were converted into p-nitrophenol concentrations (μM) to determine the initial reaction rate of each variant. Borneol dehydrogenase (BDH) activity assay: Activity was assessed by measuring the conversion of borneol (+) and NAD+ to camphor and NADH. A final concentration of 3 μM BDH was added to a 200 μL solution of 0.5 mM borneol (+) and 0.2 mM NAD+ (in 100 mM Tris, 5% v\v DMSO, pH 9) in a 96-well assay plate at 20°C. The absorbance at 340 nm was measured at 15 s intervals for 10 min, determining the formation of NADH. Temperature resistance of the enzyme was measured by incubating a 15 μM stock solution of BDH (in 50 mM potassium phosphate, 50 mM potassium chloride, pH 8) at varying temperature (20–60°C) for 15 min, before cooling to 20°C and performing the assay as described. Example 1: Synthesis of crosslinker Co2 (1,1',1''-(1,7,13-trioxa-4,10,16- triazacyclooctadecane-4,10,16-triyl)tris(2-chloroethan-1-one)) To a solution of 1,7,13-trioxa-4,10,16-triazacyclooctadecane (50 mg, 0.19 mmol) in CH2Cl2 (1 M) was added K2CO3 (118 mg, 0.86 mmol) in H2O at 0 °C. Upon vigorous stirring, 2- chloroacetyl chloride (55 µL, 0.69 mmol) was dropwise added, and the mixture was allowed to stir at room temperature overnight. The mixture was diluted with water and extracted three times with CH2Cl2. Subsequently, the organic layer was washed with brine, dried over Na2SO4, and concentrated in vacuo. The crude was further purified by silica gel column chromatography. The title compound was isolated without purification as a white amorphous solid (63 mg, 0.13 mmol, 68%).1H NMR (500 MHz, DMSO-d6) δ 4.41 (t, J = 2.2 Hz, 6H), 3.65 – 3.43 (m, 24H).13C NMR13C NMR (126 MHz, DMSO-d6) δ 166.3 (3C), 69.7 (3C), 68.3 (3C), 48.7 (3C), 47.8 (3C), 42.3 (3C). IR (neat) vmax (cm-1) = 1637, 1460, 1418, 1354, 1107, 1060, 1014. HRMS (ESI) calcd for C18H31Cl3N3O6 [M+H]+= 490.1273 found 490.1284. Example 2: Synthesis of crosslinker Co3 (4,10,16-tris(vinylsulfonyl)-1,7,13- trioxa-4,10,16-triazacyclooctadecane)) To a solution of 2-chloroethanesulfonyl chloride (89 uL, 0.86 mmol) in CH2Cl2 (1.0 M) was added triethylamine (131 µL, 0.94 mmol) at −30 °C. After being stirred for 2 h, a solution of 1,4,7-triazacyclononane trihydrochloride (50 mg, 0.19 mmol) and triethylamine (131 µL, 0.94 mmol) in CH2Cl2 (0.5 M) was dropwise added, after which the temperature was allowed to slowly increase to room temperature over 16 h. The suspension was filtered and quenched in cold HCl (0.1 M). The product was extracted with CH2Cl2, dried over Na2SO4, and concentrated in vacuo. The crude was purified by flash chromatography using EtOAc / cyclohexane (2:1) as eluent to obtain the title compound as a yellowish oil (31 mg, 0.058 mmol, 31%). Rf = 0.18 (EtOAc / cyclohexane 2:1).1H NMR (500 MHz, CDCl3) δ 6.44 (dd, J = 16.5, 9.9 Hz, 3H), 6.21 (d, J = 16.5 Hz, 3H), 5.95 (d, J = 9.9 Hz, 3H), 3.63 (t, J = 5.7 Hz, 12H), 3.37 (t, J = 5.7 Hz, 12H). 13C NMR (126 MHz, CDCl3) δ 134.2 (3C), 127.0 (3C), 70.9 (6C), 49.6 (6C). IR (neat) vmax (cm-1) = 1323, 1138, 1117, 991, 966, 953. HRMS (ESI) calcd for C18H34N3O9S3 [M+H]+= 532.1452; found, 532.1457. Example 3: Synthesis of crosslinker Cn2 (1,1',1''-(1,4,7-triazonane-1,4,7- triyl)tris(2-chloroethan-1-one)) To a solution of triamine 1,4,7-triazacyclononane trihydrochloride (239 mg, 1 mmol)) in CH2Cl2 (1 M) was added K2CO3 (1.11 g, 8 mmol) in H2O at 0 °C. Upon vigorous stirring, 2- chloroacetyl chloride (0.27 ml, 3.3 mmol) was dropwise added, and the mixture was allowed to stir at room temperature overnight. The mixture was diluted with water and extracted three times with CH2Cl2. Subsequently, the organic layer was washed with brine, dried over Na2SO4, and concentrated in vacuo. The title compound was isolated without purification as a white amorphous solid (130 mg, 0.36 mmol, 36%).1H NMR (600 MHz, DMSO-d6) δ 4.34 (s, 6H), 3.60 (s, 6H), 3.41 (s, 6H).13C NMR (151 MHz, DMSO) δ 167.3 (3C), 50.6 (3C), 48.0 (3C), 42.4 (3C). IR (neat) vmax (cm-1) = 1643, 1630, 1458, 1445, 1412, 1358, 1219, 1190. HRMS (ESI) calcd for C12H19Cl3N3O3 [M+H]+= 358.0487, found 358.0486. Example 4: Synthesis of crosslinker Cn3 (1,4,7-tris(vinylsulfonyl)-1,4,7- triazonane)) To a solution of 2-chloroethanesulfonyl chloride (470 uL, 4.5 mmol) in CH2Cl2 (1 mL) was added triethylamine (697 µL, 5 mmol) at −30 °C. After being stirred for 2 h, a solution of 1,4,7-triazacyclononane trihydrochloride (239 mg, 1 mmol) and triethylamine (975 µL, 7 mmol) in CH2Cl2 (0.5 M) was dropwise added, after which the temperature was allowed to slowly increase to room temperature over 16 h. The suspension was filtered and quenched in cold HCl (0.1 M). The product was extracted with CH2Cl2, dried over Na2SO4, and concentrated in vacuo. The crude was purified by flash chromatography using EtOAc / cyclohexane (2:1) as eluent to obtain the title compound as a yellowish oil (30 mg, 0.075 mmol, 7.5%). Rf = 0.22 (EtOAc / cyclohexane 2:1).1H NMR (600 MHz, DMSO-d6) δ 6.89 (dd, J = 16.5, 10.0 Hz, 3H), 6.14 (d, J = 10.0 Hz, 3H), 6.11 (d, J = 16.5 Hz, 3H), 3.32 (s, 12H).13C NMR (151 MHz, DMSO) δ 133.4 (3C), 129.0 (3C), 51.6 (3C). IR (neat) vmax (cm1) = 1319, 1142, 970, 959, 740. HRMS (ESI) calcd for C12H22N3O6S3 [M+H]+= 400.0665, found 400.0676. Example 5: Synthesis of crosslinker Bz4 (N,N',N''-(benzene-1,3,5- triyltris(methylene))tris(2-iodoacetamide)) A round bottom flask was charged with 100 mg of 3 (0.253 mmol, 1 equiv.) and suspended in 2.5 mL of acetone. 683 mg (4.56 mmol, 18 equiv.) of sodium iodine were added and the mixture was stirred at room temperature for 18h. The precipitated salts were filtered off and washed with 3 mL of acetone. 3 mL of water were slowly added to the flow through under stirring. A heavy white solid precipitated, was filtered off and washed with 2 x 4 mL acetone / water 1 / 1. The solid was resuspended in 4 mL of water and lyophilized to give 118 mg (0.176 mmol, LC-MS purity 99%) of 4 as a white powder in 69% yield.1H NMR (500 MHz, DMSO) δ 8.76 (t, J = 5.9 Hz, 3H), 7.06 (s, 3H), 4.25 (d, J = 5.9 Hz, 6H), 3.71 (s, 6H). HRMS (ESI) calcd. for C15H18I3N3O3 [M+H]+= 669.8555, found 669.8284. Example 6: Synthesis of crosslinker Ta4 (N,N',N''-((1,3,5-triazinane-1,3,5- triyl)tris(2-oxoethane-2,1-diyl))tris(2-iodoacetamide)) A round bottom flask was charged with 100 mg (0.206 mmol, 1 equiv.) of 9 and suspended in 2.1 mL of acetone. 559 mg (3.70 mmol, 18 equiv.) of sodium iodine were added and the reaction mixture was stirred at room temperature for 16h.2 mL of water were added and the clear yellowish solution was directly subjected to reverse phase chromatography (water +0.1% FA / ACN+0.1% FA 90 / 10 to 10 / 90 in 15 min). Pure fractions were combined and lyophilized to give 120 mg (0.157 mmol, LC-MS purity 99%) of a white lyophilizate in 76% yield.1H NMR (300 MHz, DMSO) δ 8.47 (d, J = 6.2 Hz, 3H), 5.27 (s, 6H), 4.16 (d, J = 5.3 Hz, 6H), 3.74 (s, 6H). HRMS (ESI) calcd. for C15H21I3N6O6 [M+H]+= 762.8729, found 762.8210. Example 7: Synthesis of crosslinker Ta5 (N,N',N''-((1,3,5-triazinane-1,3,5- triyl)tris(2-oxoethane-2,1-diyl))tris(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)hexanamide)) 395 mg (1.87 mmol, 3.3 equiv.) of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoic acid were dissolved in 3 mL of DMF and 345 μL of DIPEA (1.98 mmol, 3.5 equiv.).562 mg (1.87 mmol, 3.3 equiv.) of TSTU were added and the clear solution was stirred for 15 min. Subsequently, the yellowish solution was added dropwise to a suspension of 181mg (0.57 mmol, content 81%, 1 equiv.) of 7 in 3 mL of DMF and stirred at room temperature for 2h. 20 mL of 1N HCl was added and the mixture was extracted with 60 mL of ethyl acetate. The aqueous layer was drained off and re-extracted with 60 mL of ethyl acetate. The organic layers were washed with 20 mL of brine, combined, dried over Na2SO4 and evaporated to give a yellowish oil which was directly subjected to FCC (dichloromethane / MeOH, gradient from 100 / 0 to 80 / 20 in 16 min) to give 97 mg (0.12 mmol, LC-MS purity 99%) of 10 as a white lyophilizate in 20% yield.1H NMR (300 MHz, DMSO) δ 7.98 (s, 3H), 7.01 (s, 5H), 5.25 (s, 6H), 4.09 (d, J = 5.5 Hz, 6H), 3.38 (d, J = 7.1 Hz, 5H), 2.11 (t, J = 7.4 Hz, 6H), 1.55 – 1.40 (m, 13H), 1.21 (q, J = 7.4 Hz, 6H). HRMS (ESI) calcd. for C39H51N9O12 [M+H]+= 838.3730, found 838.3344. Example 8: Synthesis of crosslinker Bb2 (N,N',N''-(benzene-1,3,5- triyltris(methylene))tris(3-(2-chloroacetamido)propanamide)) To a stirred solution of trisamine.3HCl (0.547 g, 1.12 mmol) in water (2.0 mL) was added K2CO3 (0.930 g, 6.73 mmol) at rt. The mixture was cooled to 0 °C, and a solution of 2- chloroacetyl chloride (0.29 mL, 3.70 mmol) in DCM (2.0 mL) was slowly added. The mixture was stirred vigorously for 3 h while being allowed to warm to room temperature. The mixture was diluted with further DCM and water and the precipitate filtered off. The solids were washed with water and DCM and dried in vacuo to afford the title compound as a white amorphous solid (0.266 g, 0.44 mmol, 39%).1H NMR (500 MHz, DMSO) δ 8.41 (t, J = 5.9 Hz, 3H), 8.27 (t, J = 5.9 Hz, 3H), 6.99 (s, 3H), 4.23 (d, J = 5.9 Hz, 6H), 4.04 (s, 6H), 2.34 (t, J = 7.1 Hz, 6H) - CH2 signal beneath the water peak at 3.33 ppm, confirmed to be present via COSY NMR. Only signals corresponding to the major rotamer are reported.13C NMR (126 MHz, DMSO) δ 170.17, 165.86, 139.59, 124.62, 42.59, 41.95, 35.68, 34.86. HRMS calcd. for C24H34Cl3N6O6+[M+H]+= 607.1600, found 607.1599. Example 9: Synthesis of crosslinker Bb4 (N,N',N''-(benzene-1,3,5- triyltris(methylene))tris(3-(2-iodoacetamido)propanamide)) To a solution of trisamine.3HCl salt (310 mg, 0.64 mmol) in water (3.80 mL) was added K2CO3 (0.54 g, 3.88 mmol) at 0 °C and a solution of 2-iodoacetyl chloride (0.19 mL, 2.10 mmol) in DCM (2.3 mL) was slowly added. The reaction stirred vigorously for 2 h at this temperature. The mixture was diluted with water and DCM, and the precipitate filtered off. The crude product was purified using reverse-phase chromatography on C-18 silica using a gradient of 10-100% ACN / H2O (+0.1% FA) to afford the title compound (10 mg, 0.011 mmol, 2% yield) as a white solid.1H NMR (500 MHz, DMSO) δ 8.42 – 8.36 (m, 3H), 8.33–8.25 (m, 3H), 6.99 (s, 3H), 4.23 (d, J = 5.9 Hz, 6H), 3.28 (q, J = 6.8 Hz, 6H), 2.31 (t, J = 6.8 Hz, 6H). HRMS calcd. for C24H34I3N6O6+[M+H]+= 904.9488, found 904.9489. Example 10: Synthesis of crosslinker Bg2 (N,N',N''-(benzene-1,3,5- triyltris(methylene))tris(3-(2-(2-chloroacetamido)ethoxy)propanamide)) To a stirred solution of tris-amine.3TFA salt (0.273 g, 0.34 mmol) in water (2.0 mL) was added K2CO3 (0.469 g, 3.39 mmol) at room temperature. This solution was then cooled to 0 °C, followed by slow addition of a solution of 2-chloroacetyl chloride (0.13 mL, 1.70 mmol) in DCM (2.0 mL). The mixture was stirred vigorously for 18 h while being allowed to warm to rt. The mixture was diluted with further DCM and water. The layers were separated, and the organic layer extracted with water. The combined aqueous layers were concentrated in vacuo. The crude product was purified with reverse-phase chromatography on C-18 silica using a gradient of 5-95% ACN / H2O (+0.1% FA) to afford the title compound (45 mg, 0.061mmol, 18% yield) as a white solid.1H NMR (500 MHz, DMSO) δ 8.34 (t, J = 6.0 Hz, 3H), 8.23 (t, J = 5.7 Hz, 3H), 6.98 (s, 3H), 4.22 (d, J = 6.0 Hz, 6H), 4.05 (s, 6H), 3.63 (t, J = 6.5 Hz, 6H), 3.42 (t, J = 6.0 Hz, 6H), 3.24 (app. q, J = 6.0 Hz, 6H), 2.39 (t, J = 6.5 Hz, 6H).13C NMR (126 MHz, DMSO) δ 169.95, 165.97, 139.58, 124.55, 68.36, 66.54, 42.55, 41.96, 38.88, 35.95. HRMS calcd. for C30H46Cl3N6O9+[M+H]+= 739.2387, found 739.2378. Example 11: Synthesis of crosslinker Bg4 (N,N',N''-(benzene-1,3,5- triyltris(methylene))tris(3-(2-(2-iodoacetamido)ethoxy)propanamide)) To a stirred solution of tris-amine.3TFA salt (0.273 g, 0.34 mmol) in water (2.0 mL) was added K2CO3 (0.469 g, 3.39 mmol) at room temperature. This solution was then cooled to 0°C, followed by slow addition of a solution of 2-iodoacetyl chloride (0.15 mL, 1.70 mmol) in DCM (2.0 mL). The mixture was stirred vigorously for 18 h while being allowed to warm to room temperature. The mixture was diluted with further DCM and water, and a brown precipitate was filtered off and washed with further water and DCM. The crude product was purified with reverse-phase chromatography on C-18 silica using a gradient of 5-95% ACN / H2O (+0.1% FA) to afford the title compound (0.160 g, 0.14 mmol, 43% yield) as a fluffy white solid.1H NMR (500 MHz, DMSO) δ 8.34 (t, J = 5.9 Hz, 3H), 8.29 (t, J = 5.7 Hz, 3H), 4.22 (d, J = 5.9 Hz, 6H), 3.66–3.61 (m, 12H), 3.39 (t, J = 5.7 Hz, 6H), 3.19 (app. q, J = 5.7 Hz, 6H), 2.40 (t, J = 6.6 Hz, 6H) - (only signals corresponding to the major rotamer are reported).13C NMR (126 MHz, DMSO) δ 169.95, 167.71, 139.58, 124.56, 68.36, 66.61, 41.97, 39.10, 35.97, 0.76. HRMS calcd. for C30H45I3N6O9Na+[M+Na]+= 1037.0274, found 1037.0263. Example 12: Synthesis of crosslinker Tb2 (N,N',N''-((1,3,5-triazinane-1,3,5- triyl)tris(2-oxoethane-2,1-diyl))tris(3-(2-chloroacetamido)propanamide)) To a solution of trisamine (60 mg, 0.12 mmol) containing piperidine (40 mg, 0.47 mmol) in water (1 mL) was added a solution of 2-chloroacetyl chloride (0.12 mL, 1.53 mmol) in DCM (1.0 mL) at 0 °C, and the reaction stirred vigorously for 5 h at this temperature. The reaction was diluted with water and DCM, the layers separated, and the organic layer extracted with water (x2). The combined aqueous layers were concentrated in vacuo. The crude product was purified with reverse-phase chromatography on C-18 silica using a gradient of 5-100% ACN / H2O (+0.1% FA) to afford the title compound (9 mg, 0.013 mmol, 10% yield) as a sticky pale-yellow solid.1H NMR (500 MHz, MeOD) δ 5.37 (s, 6H), 4.27 (s, 6H), 4.06 (s, 6H), 3.52 (t, J = 6.6 Hz, 6H), 2.50 (t, J = 6.6 Hz, 6H).13C NMR (126 MHz, MeOD) δ 174.18, 169.98, 169.32, 43.27, 42.02, 37.35, 36.07.13C signal at 55.4 ppm confirmed by HSQC analysis. HRMS calcd. for C24H37Cl3N9O9+[M+H]+= 700.1775, found 700.1771. Example 13: Synthesis of crosslinker Tb4 (N,N',N''-((1,3,5-triazinane-1,3,5- triyl)tris(2-oxoethane-2,1-diyl))tris(3-(2-iodoacetamido)propanamide)) To a solution of trisamine.3HCL (409 mg, 0.87 mmol) containing diethylamine (127 mg, 1.49 mmol) in water (5.0 mL) was added K2CO3 (566 mg, 4.10 mmol). A solution of 2- iodoacetyl chloride (0.39 mL, 4.34 mmol) in DCM (5.0 mL) was added slowly at 0 °C and the reaction was allowed to warm to room temperature and stirred vigorously for 5 h. The reaction mixture was diluted with water and DCM, and the phases separated. The organic layer was extracted with water (x2) and the combined aqueous layers were concentrated in vacuo. The crude product was purified on reverse-phase C-18 silica using a gradient of 5-95% MeCN / water (+0.1% FA) to afford the title compound (55 mg, 0.055 mmol, 7% yield) as a sticky pale yellow solid.1H NMR (500 MHz, D2O) δ 5.40 (s, 6H), 4.31 (s, 6H), 3.76 (s, 6H), 3.50 (t, J = 6.5 Hz, 6H), 2.57 (t, J = 6.5 Hz, 6H). HRMS calcd. for C24H36I3N9O9Na+[M+Na]+= 997.9662, found 997.9664. Example 14: Synthesis of crosslinker Tg2 (N,N',N''-((1,3,5-triazinane-1,3,5- triyl)tris(2-oxoethane-2,1-diyl))tris(3-(2-(2- chloroacetamido)ethoxy)propanamide)) To a stirred solution of trisamine.3TFA salt (0.298 g, 0.33 mmol) in water (2.0 mL) was added K2CO3 (0.367 g, 2.65 mmol) at rt. This solution was then cooled to 0 °C, followed by slow addition of a solution of 2-chloroacetyl chloride (0.09 mL, 0.087 mmol) in DCM (2.0 mL). The mixture was stirred vigorously for 18 h while being allowed to warm to room temperature. The mixture was diluted with further DCM and water. The layers were separated, and the organic layer extracted with water (x2). The combined aqueous layers were concentrated in vacuo. The crude product was purified with reverse-phase chromatography on C-18 silica using a gradient of 5-95% ACN / H2O (+0.1% FA) to afford the title compound (0.070 g, 0.084 mmol, 25% yield) as colourless sticky solid.1H NMR (500 MHz, DMSO) δ 8.20 (t, J = 5.5 Hz, 3H), 8.05 (br. s, 3H), 5.26 (s, 6H), 4.13 (d, J = 5.5 Hz, 6H), 4.05 (s, 6H), 3.60 (t, J = 6.5 Hz, 6H), 3.40 (t, J = 5.7 Hz, 6H), 3.23 (app. q, J = 5.7 Hz, 6H), 2.40 (t, J = 6.5 Hz, 6H).13C NMR (126 MHz, DMSO) δ 170.35, 168.12, 165.97, 68.38, 66.43, 42.58, 40.28, 38.89, 35.72.13C signal at 55.5 ppm confirmed by HSQC analysis. HRMS calcd. for C30H48Cl3N9O12Na+[M+Na]+= 854.2380, found 854.2380. Example 15: Synthesis of crosslinker Tg4 (N,N',N''-((1,3,5-triazinane-1,3,5- triyl)tris(2-oxoethane-2,1-diyl))tris(3-(2-(2-iodoacetamido)ethoxy)- propanamide)) To a stirred solution of trisamine.3TFA salt (0.149 g, 0.17 mmol) in water (1.0 mL) was added K2CO3 (0.183 g, 1.32 mmol) at room temperature. This solution was then cooled to 0 °C, followed by slow addition of a solution of 2-iodoacetyl chloride (0.05 mL, 0.55 mmol) in DCM (1.0 mL). The mixture was stirred vigorously for 18 h while being allowed to warm to rt. The mixture was diluted with further DCM and water. The layers were separated, and the organic layer extracted with water (x2). The combined aqueous layers were concentrated in vacuo. The crude product was purified with reverse-phase chromatography on C-18 silica using a gradient of 5-95% ACN / H2O (+0.1% FA) to afford the title compound (39 mg, 0.035 mmol, 21% yield) as a sticky off-white solid.1H NMR (500 MHz, DMSO) δ 8.26 (t, J = 5.6 Hz, 3H), 8.05 (br. s, 3H), 5.26 (s, 7H), 4.13 (d, J = 5.6 Hz, 6H), 3.64 (s, 6H), 3.60 (t, J = 6.5 Hz, 7H), 3.38 (t, J = 5.7 Hz, 6H), 3.19 (app. q, J = 5.7 Hz, 6H), 2.41 (t, J = 6.5 Hz, 6H).13C NMR (126 MHz, DMSO) δ 170.34, 167.70, 167.11, 68.38, 66.49, 40.30, 39.09, 35.73, 0.82.13C signal at 55.6 ppm confirmed by COSY analysis. HRMS calcd. for C30H48I3N9O12Na+[M+Na]+= 1130.0449, found 1130.0437. Example 16: Synthesis of crosslinker Ta-DBCO (N1,N1',N1''-((1,3,5-triazinane- 1,3,5-triyl)tris(2-oxoethane-2,1-diyl))tris(N6,N6-dicyclobenzoazacyclo- octynylamide) To a suspension of benzoazacyclooctyne-carboxylic acid (DBCO-COOH) (10 mg, 0.033 mmol) in THF (2.2 mL) were added three drops of DMF. The reaction mixture was cooled to 0 °C before addition of DIPEA (0.009 mL, 0.050 mmol), HATU (16.3 mg, 0.043 mmol) and trisamine 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)tris(2-aminoethan-1-one) (2.56 mg, 0.010 mmol). The reaction mixture was stirred for 18 hours while being allowed to warm to room temperature. The volatiles were removed in vacuo and the residue partitioned between 1 M HCl and EtOAc, and the aqueous extracted with EtOAc (x3). The combined organics were dried over Na2SO4 and concentrated in vacuo. The crude product was purified by reverse phase semi-preparative liquid chromatography to afford the title compound solid (0.6 mg, 0.050 mmol, 5%) as a white amorphous solid.1H NMR (600 MHz, DMSO) δ 7.61 (d, J = 7.1 Hz, 3H), 7.59–7.25 (m, 21H), 5.18 (s, 6H), 5.03 (d, J = 14.0 Hz, 3H), 3.99 (s, 6H), 3.60 (d, J = 13.9 Hz, 3H), 1.99 (ddd, J = 14.2, 7.2, 6.9 Hz, 6H), 1.90 (t, J = 6.4 Hz, 6H), 1.21 – 1.13 (m, 12H). HRMS calcd. for C72H69N9O9Na+[M+Na]+= 1226.5110, found 1226.5113. Example 17: Synthesis of crosslinker Ap-DBCO (N1,N1',N1''- (nitrilotris(propane-3,1-diyl))tris(N6,N6-dicyclobenzoazacyclooctynylamide)) To a suspension of dibenzoazacyclooctyne-carboxylic acid (DBCO-COOH) (30 mg, 0.090 mmol) in DCM (1.8 mL) and THF (0.4 mL) was added two drops of DMF at room temperature. The reaction mixture was cooled to 0 °C and DIPEA (0.024 mL, 0.135 mmol), HATU (44.5 mg, 0.117 mmol) and tris(3-aminopropyl)amine (0.005 mL, 0.027 mmol) were added and the mixture was stirred for 18 h while allowing to warm to room temperature. The mixture was diluted with a solution of 1 M HCl and EtOAc. The organic layer was collected, dried over Na2SO4 and concentrated in vacuo. The crude product was purified using reverse-phase chromatography using a gradient of 5-95% ACN / H2O (+0.1% FA) to afford the title compound (8 mg, 0.007 mmol, 26% yield) as a white amorphous solid.1H NMR (500 MHz, DMSO) δ 7.76 (t, J = 5.9 Hz, 3H), 7.61 (d, J = 7.4 Hz, 3H), 7.58 – 7.24 (m, 21H), 5.02 (d, J = 14.1 Hz, 3H), 3.60 (d, J = 14.1 Hz, 3H), 2.99 (m, 6H), 2.96 – 2.88 (m, 6H), 2.16 (ddd, J = 14.6, 8.2, 5.5 Hz, 3H), 1.84 (t, J = 6.9 Hz, 6H), 1.72 (ddd, J = 14.9, 8.6, 5.8 Hz, 3H), 1.63 (tt, J = 8.2, 7.5 Hz, 6H), 1.32–1.11 (m, 12H).13C NMR (126 MHz, DMSO) δ 172.18, 171.64, 151.77, 148.46, 132.39, 129.44, 128.88, 128.13, 128.00, 127.67, 126.79, 125.14, 122.47, 121.40, 114.34, 108.17, 54.80, 49.99, 35.52, 34.95, 33.95, 24.54, 23.64. HRMS calcd. for C72H76N7O6+[M+H]+= 1134.5850, found 1134.5854. Example 18: Synthesis of crosslinker IA-C3 (2-iodo-N-(prop-2-yn-1-yl)acetamide) Precursor chloroacetamide (500 mg, 3.81 mmol, 1 equiv.) is suspended in acetone (38 mL) acetone. After the addition of NaI (1.7 g, 11.34 mmol, 3 equiv.), the reaction mixture is stirred at room temperature for 16 h. The reaction mixture is concentrated in vacuo and purified by manual column chromatography (isocratic, ethyl acetate / cyclohexane 50:50) to give 729 mg (3.27 mmol) of the title compound as a white solid in 91% yield.1H NMR (300 MHz, CDCl3) δ 6.35–6.28 (m, 1H), 4.10 (dd, J = 5.4, 2.4 Hz, 3H), 3.74 (s, 2H), 2.30 (td, J = 2.6, 0.8 Hz, 1H). Example 19: Synthesis of crosslinker IA-C5 (2-iodo-N-(pent-4-yn-1-yl)acetamide) Precursor chloroacetamide (200 mg, 1.26 mmol, 1 equiv.) is suspended in acetone (13 mL). After the addition of NaI (565 mg, 3.77 mmol, 3 equiv.), the reaction mixture is stiffed at room temperature for 16h. The reaction mixture was concentrated in vacuo and purified by manual column chromatography (isocratic, ethyl acetate / cyclohexane 50:50) to give 119 mg (3.27 mmol) of a colorless oil which was redissolved in ACN / water (1:1) and lyophilized to obtain 114 mg (0.454 mmol) of the title compound as a white lyophilizate in 33% yield over 2 steps.1H NMR (300 MHz, CDCl3) δ 6.32 (s, 1H), 3.72 (s, 2H), 3.43 (q, J = 6.6 Hz, 2H), 2.30 (td, J = 6.9, 2.7 Hz, 2H), 2.08–2.00 (m, 2H), 1.79 (p, J = 6.8 Hz, 2H). Example 20: Crosslinking PFE variants (Fig. 4) Pseudomonas fluorescens aryl esterase (PFE) is reported to be present as a homotrimer (PDB ID: 1va4). PFE-T2, PFE-Q173 were designed to carry one cysteine variation per monomer to allow the reaction with one trivalent crosslinker Ta4 to give a covalently linked trimer. PFE-DM is a combination of PFE-T2 and PFE-Q173 carrying two cysteine variations per monomer to allow the reaction with two crosslinkers Ta4 to provide a doubly crosslinked trimer. See Figure 4 for crystal structure of the unmodified protein complex with indicated crosslinking sites and analytical data. The table below shows the stabilization effect of the different crosslinked variants. Protein Variant Multimer No. of MW MW ^Ti crosslinkers calcd. obs. (°C) per trimer PFE-T2-Ta T2C homotrimer 1 93171 93161 1.6 PFE-Q173-Ta Q173C homotrimer 1 93090 93079 4.7 PFE-DM-Ta2 T2C, Q173C homotrimer 2 93478 93460 5.6 * ^Ti (°C): Difference in inflection temperature compared to PFE-wt (no cysteine variation and no crosslinking). PFE-T2 Sequence (SEQ ID NO:1): SCFVAKDGTQIYFKDWGSGKPVLFSHGWLLDADMWEYQMEYLSSRGYRTIAFDRRG FGRSDQPWTGNDYDTFADDIAQLIEHLDLKEVTLVGFSMGGGDVARYIARHGSARVA GLVLLGAVTPLFGQKPDYPQGVPLDVFARFKTELLKDRAQFISDFNAPFYGINKGQVV SQGVQTQTLQIALLASLKATVDCVTAFAETDFRPDMAKIDVPTLVIHGDGDQIVPFET TGKVAAELIKGAELKVYKDAPHGFAVTHAQQLNEDLLAFLKRGSHHHHHH PFE-Q173 Sequence (SEQ ID NO:2): STFVAKDGTQIYFKDWGSGKPVLFSHGWLLDADMWEYQMEYLSSRGYRTIAFDRRG FGRSDQPWTGNDYDTFADDIAQLIEHLDLKEVTLVGFSMGGGDVARYIARHGSARVA GLVLLGAVTPLFGQKPDYPQGVPLDVFARFKTELLKDRAQFISDFNAPFYGINKGQVV SCGVQTQTLQIALLASLKATVDCVTAFAETDFRPDMAKIDVPTLVIHGDGDQIVPFET TGKVAAELIKGAELKVYKDAPHGFAVTHAQQLNEDLLAFLKRGSHHHHHH PFE-DM Sequence (SEQ ID NO:3): SCFVAKDGTQIYFKDWGSGKPVLFSHGWLLDADMWEYQMEYLSSRGYRTIAFDRRG FGRSDQPWTGNDYDTFADDIAQLIEHLDLKEVTLVGFSMGGGDVARYIARHGSARVA GLVLLGAVTPLFGQKPDYPQGVPLDVFARFKTELLKDRAQFISDFNAPFYGINKGQVV SCGVQTQTLQIALLASLKATVDCVTAFAETDFRPDMAKIDVPTLVIHGDGDQIVPFET TGKVAAELIKGAELKVYKDAPHGFAVTHAQQLNEDLLAFLKRGSHHHHHH PFE-wt Sequence (SEQ ID NO:4): STFVAKDGTQIYFKDWGSGKPVLFSHGWLLDADMWEYQMEYLSSRGYRTIAFDRRG FGRSDQPWTGNDYDTFADDIAQLIEHLDLKEVTLVGFSMGGGDVARYIARHGSARVA GLVLLGAVTPLFGQKPDYPQGVPLDVFARFKTELLKDRAQFISDFNAPFYGINKGQVV SQGVQTQTLQIALLASLKATVDCVTAFAETDFRPDMAKIDVPTLVIHGDGDQIVPFET TGKVAAELIKGAELKVYKDAPHGFAVTHAQQLNEDLLAFLKRGSHHHHHH Example 21: Two-component crosslinking of PFE variants (Fig.5) Step 1: Step 2: Two-component crosslinking with different iodoacetamide handles and different core structures. For alkylating with IA-C3 / 5, PFE variants (50μM of monomer) were incubated with 1mM IA-C3 / 5in crosslinking buffer for 2-3 h at 40 °C and 200 rpm. Samples were taken and analyzed via LC-MS. A buffer exchanged to clicking buffer and concentration step was performed using spin filter columns. For the crosslinking step of alkylated PFE versions, these were diluted in crosslinking buffer (45 μM of monomer final). Depending on the variant, Ta- or Bz-azide (Q173C = 30 μM, T2C = 40 μM, DM = 80 μM) was added. Subsequently, premixed CuSO4 (100 μM) with THPTA ligand (500 μM) were added. The reaction was initiated through addition of sodium ascorbate (5 mM) and incubated for 3 h at 40 °C and 200 rpm. See Figure 5 for analytical data. Esterase activity assay: Protein variants were heated for 1 h at indicated temperatures and then cooled to 4 °C using an Eppendorf Mastercycler® pro. Example 22: Crosslinking of four homotrimeric proteins (Fig. 6 – 9) Rhodanese domain-containing protein (I4) from Lactiplantibacillus plantarum, Enoyl- CoA hydratase / isomerase family protein (e4) from Hyphomonas neptunium, Tautomerase_3 domain-containing protein (a4) from Neosartorya fumigate, BH3498 protein (b4) from Halalkalibacterium halodurans are each reported to exist as homotrimers (PDB IDs: 3fnj, 5c9g, 3c6v, 1vmf, respectively). For each protein complex a cysteine variant was designed that allows double crosslinking of the homotrimer complex in analogy to PFE-DM-Ta2, resulting in the stabilized complexes as shown in the table below. See Figures 6 – 9 for crystal structures of the unmodified protein complexes and analytical data. Protein Variant Native Cross- No. of MW MW obs. ^TM state linked Ta per calcd. (°C)* dimer I43-Ta2 E7C, E108C trimer trimer 2 43421 43422 19 e43-Ta2 S198C, G243C trimer trimer 2 90610 90607 17 a43-Ta2 E66C, A91C trimer trimer 2 55992 55994 39 b43-Ta2 E78C, T144C trimer trimer 2 49901 49903 6 * ^TM(°C): Difference in melting temperature toward non-crosslinked protein. I4 Sequence (SEQ ID NO:5): MNDKKICLLTTYLSLYIDHHTVLADMQNATGKYVVLDVRNAPAQVKKDQIKGAIAMP AKDLATRIGELDPAKTYVVYDWTGGTTLGKTALLVLLSAGFEAYELAGALCGWKGM QLPVETLADLEHHHHHH e4 Sequence (SEQ ID NO:6): MHHHHHHSSGVDLGTENLYFQSMTLPIRLDIAAPLAEIVLNKPERRNALSVDMWAAI PGLVAEANANPDVKLILIHGGDAGAFAAGADISEFETIYATEDAAKASGQRIAQALDAI ENSEKPVIAAIEGACVGGGVSLAMAADLRVAGEGAKFGVTPGKLGLVYPAGDTRRLL AAVGPGATKDILFTGRIFTAGEAKCLGLIDRLVEKGTALEAARVWAGEIAAISQWSVR ATKRMIRGLQTCWTDETPEAQSLFLNGFANEDFKEGYRAFLDKRPAKFTYR a4 Sequence (SEQ ID NO:7): MGSSHHHHHHSSGRENLYFQGMPRWLIQHSPNTLTPEEKSHLAQQITQAYVGFGLP AFYVQVHFICQPAGTSFIGGEQHPNFVALTIYHLCRTMTSDEQRQGFLKRIDAFLTPM FEPKGIDWEYFVTEAPRDLWKINGLAPPAAGSEEEKVWVRENRPVRF b4 Sequence (SEQ ID NO:8): MGSDKIHHHHHHMKTFHLTTQSRDEMVDITSQIETWIRETGVTNGVAIVSSLHTTAG ITVNENADPDVKRDMIMRLDCVYPWHHENDRHMEGNTAAHLKTSTVGHAQTLIISE GRLVLGTWQGVYFCEFDGPRTNRKFVVKLLCD Example 23: Crosslinking of Asparaginase (Fig. 10) E. coli asparaginase II is reported to exists as a homotetramer (dimer of dimers, PDB ID: 3eca). EcAsp-1 variant was designed to allow the crosslinking of each of two dimers of the tetramer with two crosslinkers Ta4 via the incorporated cysteine residues, resulting in a stabilized complex as shown in the table below. See Figure 10, for crystal structure of the unmodified protein complex and analytical data. Protein Variant Native Cross- No. of MW MW ^TM state linked Ta per calcd. obs. °C dimer EcAsp-1-Ta2 K186C,Q280C, tetramer dimer 2 70038 70039 16.4 Q321C * ^TM (°C): Difference in melting temperature compared to unmodified protein (no cysteine variation and crosslinking). EcAsp-1 Sequence (SEQ ID NO:9): GPLPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNI GSQDMNDNVWLTLAKKINTDSDKTDGFVITHGTDTMEETAYFLDLTVKSDKPVVMV GAMRPSTSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDV ATFKSVNYGPLGYIHNGCIDYQRTPARKHTSDTPFDVSKLNELPKVGIVYNYANASDL PAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTCDAE VDDAKYGFVASGTLNPQKARVLLQLALTQTKDPQQIQCIFNQY EcAsp-wt Sequence (SEQ ID NO:10): GPLPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNI GSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMV GAMRPSTSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDV ATFKSVNYGPLGYIHNGKIDYQRTPARKHTSDTPFDVSKLNELPKVGIVYNYANASDL PAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAE VDDAKYGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY Fig. 17 (C) EcAsp-1-Ta2 shows onset of aggregation at higher temperatures compared with EcAsp-wt-wt. Example 24: Crosslinking of Borneol Dehydrogenase (Fig. 11) Borneol dehydrogenase of Pseudomonas sp. TCU-HL1 is reported to exist as a homotetramer (dimer of dimers, PDB ID: 6m5n). BDH-X2 variant was designed to allow crosslinking of each of the two dimers of the tetramer with two crosslinkers Ta4 via incorporated cysteine residues, resulting in a stabilized complex as shown in the table below. See Figure 11 for crystal structure of the unmodified protein complex and analytical data, including higher activity of the stabilized version compared to unmodified at higher temperatures. Protein Variant Native Cross- No. of Ta MW MW ^Ti state linked per dimer calcd. obs. °C BDH-X22-Ta2E73C,A103C,R tetramer dimer 2 60183 60185 14.0 129C * ^Ti (°C): Difference in inflection temperature compared to unmodified protein (no cysteine variation and crosslinking). BDH-X2 Sequence (SEQ ID NO:11): MKLLEGKRIIVTGGAQGIGASVVRAYIAAGATVASMDMNDTLGQQVVSEAGKANPGS KSRYYHCNIADRPEVCKAFATAAEDMGGLDVMVNVAGVHRHSPPDCIAEELYDMLF RVNVLGTINTNAVAYCLMKGQGIGNIINFGSESGLTGEINNALYSATKAAVHTWTRN VARQWGPDGIRINAVLPYMVTPMYVDFRNALSSEDLAAHDAATKTDIPLGGKFGDAD KDLAPVMVFLASDASHFMTGQMFPVDGGLIAVR BDH-wt Sequence (SEQ ID NO:12): MKLLEGKRIIVTGGAQGIGASVVRAYIAAGATVASMDMNDTLGQQVVSEAGKANPGC KSRYYHCNIADRPEVEKAFATAAEDMGGLDVMVNVAGVHRHSPPDAIAEELYDMLF RVNVLGTINTNAVAYRLMKGQGIGNIINFGSESGLTGEINNALYSATKAAVHTWTRN VARQWGPDGIRINAVLPYMVTPMYVDFRNALSSEDLAAHDAATKTDIPLGGKFGDAD KDLAPVMVFLASDASHFMTGQMFPVDGGLIAVR Fig. 17 (B) BDH-X22-Ta2 shows onset of aggregation at higher temperatures compared with BGH-wt. Example 25: Crosslinking of Gal1 (Fig. 12) Human galectin-1 is reported to exist as a homodimer (PDB ID: 1gzw). Gal1-1 variant was designed to allow crosslinking of each individual monomer with one crosslinker Ta4, while Gal1-3 variant was designed to allow attachment of two crosslinkers Ta4 bridging the two monomers of the dimer. Crosslinking of both variants resulted in stabilized complexes as shown in the table below. See Figure 12 for crystal structure of the unmodified protein complex and analytical data. Protein Variant Native Cross- No. of MW MW ^TM state linked Ta calcd. obs. °C Gal1-1-Ta K37C,D65C,N114C Dimer Monomer 1 17992 17993 18.8 18169§18171§Gal1-3-Ta2 S3C,N11C,K128C Dimer Dimer 2 36040 36041 18.2 36217§36219§* ^TM (°C): Difference in melting temperature compared to unmodified protein (no cysteine variation and crosslinking). § MW with incl. N-terminal fMet. Gal1-1 Sequence (SEQ ID NO:13): GPMASGLVASNLNLKPGESLRVRGEVAPDAKSFVLNLGCDSNNLCLHFNPRFNAHG DANTIVCNSKCGGAWGTEQREAVFPFQPGSVAEVSITFDQANLTVKLPDGYEFKFPN RLCLEAINYMAADGDFKIKSVAFD Gal1-3 Sequence (SEQ ID NO:14): GPMACGLVASNLCLKPGESLRVRGEVAPDAKSFVLNLGKDSNNLCLHFNPRFNAHG DANTIVCNSKDGGAWGTEQREAVFPFQPGSVAEVSITFDQANLTVKLPDGYEFKFPN RLNLEAINYMAADGDFCIKSVAFD Gal1-wt Sequence (SEQ ID NO:15): GPMASGLVASNLNLKPGESLRVRGEVAPDAKSFVLNLGKDSNNLCLHFNPRFNAHG DANTIVCNSKDGGAWGTEQREAVFPFQPGSVAEVSITFDQANLTVKLPDGYEFKFPN RLNLEAINYMAADGDFKIKSVAFD Fig. 16 shows (A) the lack of aggregation of Gal1-3-Ta2 compared to Gal1-wt and (B) the full dimer state for Gal1-3-Ta2 compared to a monomer-dimer equilibrium for Gal1-wt. Example 26: Long-term temperature resistance of PFE-DM-Ta2 (Fig. 13) Hydrolysis activity of PFE variants PFE-wt and bicyclic PFE-DM-Ta2 shows retained activity of the crosslinked protein after more than three weeks of incubation at 50°C in 50 mM HEPES, 50 mM NaCl, pH 8. Example 27: Prolonged activity of EcAsp-1-Ta2 (Fig. 14) Activity measurements were performed with a commercially available asparaginase activity assay according to manufacturer’s protocol using 2 nM enzyme and measuring absorbance at 570 nm. Fig. 14 shows (A) residual activity after pre-incubation of enzymes in PBS pH 7.4 for 5 min at 25 or 65 °C and (B) activity after storage in PBS pH 7.4 for indicated times. Example 28: Ta4 crosslinked PFE (PFE-DM-Ta2) resists aggregation under thermal stress (Fig. 15)Dynamic light scattering data was recorded using a Panta Prometheus instrument at a temperature ramp rate of 1 °C / min, measured at a trimer concentration of 15 µM in 50 mM HEPES, 50 mM NaCl, pH 8. Size distributions and cumulant radii were determined using Panta Control (version 1.6.3) software. Size distribution plots represent an average of six measurements within a +0.95 °C temperature range. (A) shows calculated size distributions of PFE-wt and PFE-DM-Ta2 particles at given temperatures between 40 to 69°C, determined by dynamic light scattering (DLS). At low temperature (≤ 40°C) both structures form mono-disperse particles with a radius of approximately 4 nm. At higher temperature (50-60°C) PFE-wt is poly-disperse forming aggregates with radius of 30-60 nm, whereas PFE-DM-Ta2 remains mono-disperse. At 69°C and above PFE-wt forms large (> 1 µM) aggregates and precipitates. (B) depicts crystal structure of PFE (PDB = 1va4) showing expected radii of a trimeric complex. (C) depicts cumulant radius analysis of PFE DLS data shows predominant particle radius between 20 to 90°C, demonstrating aggregation resistance of PFE-DM-Ta2. Fig. 17 PFE-DM-Ta2 shows onset of aggregation at higher temperatures compared with PFE-wt.

Claims

Claims 1. Protein complex comprising or consisting of two or more monomers in native tertiary and / or quaternary structure which is fixed by a crosslinker having a core structure Q and at least three moieties B covalently linked to reactive groups A in one or more of the monomers forming a linkage F, wherein group A is independently a sulfhydryl group or a bioorthogonal group, and moiety B is independently a sulfhydryl-reactive group or a bioorthogonal group.

2. Protein complex according to claim 1 wherein A is a sulfhydryl group and B is a sulfhydryl-reactive group, or wherein A is a bioorthogonal group and B is a bioorthogonal group.

3. Protein complex according to claim 1 or 2 wherein the linkage F is selected from thewhereinR is an alkyl, R1is independently selected from H, F, -O(CH2)(CO)OH, -O(CH2)-pBzOH, R2is independently selected from H, -CH3, -(C6H6)CH2COOH, -pyridyl, and R3is independently selected from H, -CH3.

4. Protein complex according to any one of claims 1 to 3 wherein each moiety B of the crosslinker is covalently linked to a different reactive group A of a different monomer, wherein all of the at least three moieties (B) of a crosslinker are covalently linked to three or more reactive groups (A) of one monomer, wherein one or more of the at least three moieties (B) of the crosslinker are covalently linked to one or more reactive groups (A) of one monomer, and one or more of the at least three moieties (B) are covalently linked to one or more reactive groups (A) of one or more other monomers, or a combination thereof.

5. Protein complex according to any one of claims 1 to 4 wherein the protein complex is multi-stabilized comprising two or more monomers covalently linked to two or more crosslinkers.

6. Protein complex according to any one of claims 1 to 5 wherein the protein complex comprises or consists of at least two monomers, of at least three monomers, or of at least four monomers.

7. Protein complex according to any one of claims 1 to 6 wherein Q is selected from the group consisting ofor a combination thereof,wherein n is an integer of 1, 2, 3, 4 or 5, wherein a dashed line indicates a site where Q is directly bound to B or optionally via L.

8. Protein complex according to any one of claims 1 to 7 wherein optional L is selected from the group consisting ofwherein X is selected from NH or O, Y is selected from NH or O, Z is selected from NH or O, m is an integer of 1, 2, 3, 4 or 5, p is an integer of 1, 2, 3, 4 or 5, o is an interger in the range of 1, 2, 3 or 4, wherein the dashed line indicates a site where L is bound to Q or B.

9. Protein complex according to any one of clams 1 to 8 wherein B is selected from the group consisting ofwherein Z is selected from F, Cl, Br, I, Tos (O−SO2−C6H4−CH3), and Mes (O−SO2−CH3), R1is selected from H, F, -O(CH2)(CO)OH, -O(CH2)-pBzOH, R2is selected from H, -CH3, -(C6H6)CH2COOH, -pyridyl, R3is selected from H, -CH3, wherein each dashed indicates a site where B is bound to the linker L.

10. Protein complex according to any one of claims 1 to 9 wherein Q is selected from the group consisting ofwherein n is an integer of 1, 2, 3, 4 or 5, wherein each dashed line in Q indicats a site where Q is directly bound to B or optionally via L; L is optionally selected from the group consisting of, or a combination thereofwherein X is selected from NH or O, Y is selected from NH or O, Z is selectedfrom NH or O, m is an integer of 1, 2, 3, 4 or 5, p is an integer of 1, 2, 3, 4 or 5, o is an interger in the range of 1, 2, 3 or 4, wherein the dashed line indicates a site where L is bound to Q or B; and B is selected from the group consisting ofcombination thereof, wherein Z is selected from F, Cl, Br, I, Tos (O−SO2−C6H4−CH3), and Mes (O−SO2−CH3), wherein each dashed line in B indicates a site where B is optionally bound to L or directly to Q.

11. Protein complex according to any one of claims 1 to 10 wherein the protein complex is an enzyme, a cytokine, a chemokine, a growth factor, a hormone, an antibody, a receptor, an antigen, structural protein, a defensive protein, a storage protein, a transport protein, a contractile, a signaling, a membrane, a secreted protein or combinations thereof.

12. Method for preparing a protein complex according to any one of claims 1 to 11 comprising or consisting of the steps: a) providing a protein complex in its tertiary and / or quaternary structure, wherein one monomer comprises at least three cysteine sulfhydryl groups A and / or bioorthogonal groups A, b) optionally performing a reaction with a connector to add a bioorthogonal group to one or more of the at least three cysteine residues of the monomer, and c) contacting the monomer with a crosslinker comprising a core Q bound to at least three reactive moieties (B) such that each moiety forms a linkage (F) with one of the at least three cysteine residues or bioorthogonal group(s) of the monomer; a) providing a protein complex in its tertiary and / or quaternary structure, wherein at least three monomers comprise a sulfhydryl group A and / or a bioorthogonal group A, b) optionally performing a reaction with a connector to add a bioorthogonal group to one or more of the at least three cysteine residues of the monomer and c) contacting the at least three monomers with a crosslinker comprising a core Q bound to at least three moieties (B) such that each moiety forms a linkage (F) with the cysteine residue or bioorthogonal group of each of the at least three monomers; a) providing a protein complex in its tertiary and / or quaternary structure, wherein at least two monomers, each comprise at least one cysteine sulfhydryl group A and / or bioorthogonal group A,b) optionally performing a reaction with a connector to add a bioorthogonal group to one or more of the at least three cysteine residues of the monomers, and c) contacting the at least two monomers with a crosslinker comprising a core Q bound to at least three moieties (B) such that each moiety forms a linkage (F) with the cysteine residue or bioorthogonal group of each of the at least two monomers; or a combination thereof.

13. Method according to claim 12 wherein the connector comprises or consists of a bioorthogonal group A´, a component L and a moiety B reacting with the reactive group A of the monomer.

14. Method according to claims 12 or 13 wherein a sulfhydryl group A is introduced in the monomer which reacts with an electrophilic group of the connector comprising the bioorthogonal group to introduce the bioorthogonal group A in the monomer.

15. Method according to any one of claims 12 to 14 wherein the sulfhydryl-reactive moiety B of the crosslinker forms a covalent linkage with the sulfhydryl group A of the protein complex, wherein the bioorthogonal moiety B of the crosslinker forms a covalent linkage with the bioorthogonal group A of the protein complex, or a combination thereof in one or more monomers of the protein complex.

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