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In Biochemistry, the term “multivalency” defines a concept of simultaneous, multiple recognition/binding events between two (macro)molecular counterparts. Enabling enhanced binding strength even in the case of intrinsically moderate-potent ligands, this concept has found rather wide application in biomolecular engineering and drug design. In the present work, focused on the development of multivalent modulators of biological and biotechnological processes, oligomerization of functional biomolecules was achieved by their genetic fusion or enzyme-mediated ligation to oligomerization domains, as well as by non-covalent coiled-coil interactions of modified proteins with multivalent bioparticles. Summarized in three peer-reviewed publications given in the cumulative part, the results of this doctoral research contribute to the toolbox of synthetic and biotechnological methods for the generation of multivalent architectures with tailor-made properties. Conceptually, the study can be separated into two independent research branches, the first one exploring effects of avidity by scaffold-based oligomerization of therapeutically relevant target-binding molecules, and the second one dealing with immobilization of orthogonal biocatalytic cascades on bioparticles. In our previous investigation it was shown that engineered cystine-knot miniproteins based on the scaffold of trypsin inhibitor McoTI-II from the squash plant Momordica cochinchinensis are able to bind a therapeutically relevant target, namely cytotoxic T lymphocyte antigen 4 (CTLA-4), however, with low affinity. The potency of these binders can be improved either applying rather sophisticated and time-consuming affinity maturation, or by inducing avidity effects upon multimerization. The latter approach was considered in the present work due to the fact that dimeric CTLA 4 protein is presented on the cell surface in high copy numbers. Within the frame of this work, binding molecules of peptidic nature, among them particular oligopeptides and cystine-knot miniproteins, were attached to oligovalent scaffold proteins by genetic fusion yielding stable oligomers upon recombinant expression. As expected, oligomerization of low-affinity CTLA-4 binder cystine knot MC-CT-010 (Kd = 3.7 µM) on the Fc part of human IgG and the C-terminal oligomerization domain of human C4b binding protein (C4BP) lead to a significant improvement of its functional binding affinity. Indeed, a more than 400-fold improved Kd of 8 nM was determined for the heptavalent fusion construct comprising the C4BP scaffold and MC-CT-010 binder. In order to extend the repertoire of oligomerization methods and to ensure tailoring of multivalent architectures in a modular way, enzyme-catalyzed conjugation of functional molecules with the desired oligovalent scaffolds was applied. To this end, the Fc and C4BP scaffolds were N- or/and C-terminally functionalized with peptidic recognition tags enabling subsequent sortase A-catalyzed ligation with the ligands of interest bearing a respective counterpart. This Lego®-like strategy allowed for the fast enzyme-promoted conjugation of functional monomers at the desired positions within the scaffold. Being applied to death receptor 5 (DR5) targeting peptides (DR5TPs) and the modified Fc and C4BP scaffolds, this approach yielded dimeric, tetrameric and heptameric constructs possessing improved binding capacity towards DR5. These results are of special value as DR5 is overexpressed on cancer cells and, being crosslinked, induces an apoptotic signaling cascade. Interestingly, the strongest binding to DR5 in vitro was observed when the DR5TP ligand was attached to the carboxytermini of C4BP in a linear fashion. Furthermore, this engineered heptad revealed a remarkable biological activity, being able to specifically induce apoptosis in living COLO205 cancer cells (EC50 = 3 nM). We ascertained that ligand number per scaffold molecule as well as their position and spatial orientation is crucial for the biological activity of DR5-targeting oligomers. In general, the established platform allowed for the fast oligomerization of functional probes with further investigation of the steric factors, as well as issues of ligand density, which can influence binding and bioactivity. In addition to covalently bound oligomeric constructs, a non-covalent coiled-coil interaction was used to fabricate enzyme-loaded bioparticles able to promote orthogonal biocatalytic cascades. The carrier particles were derived from a recombinant polyhydroxyalkanoate synthase (PhaC) fusion protein, displaying a multitude of negatively charged Ecoil helices on their surface. Immobilization of enantioselective NADH-dependent alcohol dehydrogenase from Rhodococcus erythropolis and a formate dehydrogenase from Candida boidinii was achieved through the interaction of their engineered Kcoil domains with the respective Ecoil counterpart on the surface of PhaC particles. The resulting multimeric, multifunctional system enabled a catalytic cascade for the stereoselective production of chiral alcohols from ketones – an important step in the manufacturing of pharmaceuticals and fine chemicals. In the frame of our study, the presence of immobilized proteins on PhaC particles was revealed by atomic force microscopy imaging, and the resulting system appeared fully functional. Thus, complete conversion of p-chloroacetophenone to (S)-4-chloro-α-methylbenzyl alcohol by ADH with parallel cofactor regeneration by FDH was confirmed by GC-MS analysis of the reaction products. Moreover, the enantioselectivity of ADH was not affected by the immobilization onto the particles, as was confirmed by GC-MS analysis applying the chiral stationary phase (ee > 99 %). Interestingly, application of an aqueous biphasic system allowed us to use highly concentrated solutions of hydrophobic substrates in organic solvents. In addition, this multienzyme construct could be easily tailored depending on the functional task as the active components could be presented on bioparticles in different ratios, and their orientation can be controlled by the position of the coiled-coil components. |
