Dominik Schumacher

Another great example of the Tub-tag® technology for protein-protein ligation.

Abstract:
We introduce a chemoenzymatic strategy for straightforward in vitro generation of C-terminally linked fusion proteins. Tubulin tyrosine ligase is used for the incorporation of complementary click chemistry handles facilitating subsequent formation of functional bispecific antibody-fragments. This simple strategy may serve as central conjugation hub for a modular protein ligation platform.

We describe a new technique in protein synthesis that extends the existing repertoire of methods for protein modification: A chemoselective reaction that induces reactivity for a subsequent bioconjugation. Hereby, an azide building block reacts first with an ethynylphosphonite via the Staudinger‐phosphonite reaction (SPhR) to an ethynylphosphonamidate. The resulting electron‐deficient triple bond subsequently undergoes a cysteine‐selective reaction with proteins or antibodies. We demonstrate… Mehr anzeigen

We describe a new technique in protein synthesis that extends the existing repertoire of methods for protein modification: A chemoselective reaction that induces reactivity for a subsequent bioconjugation. Hereby, an azide building block reacts first with an ethynylphosphonite via the Staudinger‐phosphonite reaction (SPhR) to an ethynylphosphonamidate. The resulting electron‐deficient triple bond subsequently undergoes a cysteine‐selective reaction with proteins or antibodies. We demonstrate that ethynylphosphonamidates hold excellent cysteine‐selective reactivity combined with a superior stability of the thiol adducts. This turns our technique into a versatile and powerful tool for the facile construction of stable, functional protein conjugates. Weniger anzeigen

Peptide-MHC (pMHC) multimers have become a valuable tool for immunological research, clinical immune monitoring, and immunotherapeutic applications. Biotinylated tetramers, reversible Streptamers, or dye-conjugated pMHC multimers are distinct pMHC reagents tailored for T cell identification, traceless T cell isolation, or TCR characterization, respectively. The specific applicability of each pMHC-based reagent is made possible either through conjugation of probes or reversible multimerization… Mehr anzeigen

Peptide-MHC (pMHC) multimers have become a valuable tool for immunological research, clinical immune monitoring, and immunotherapeutic applications. Biotinylated tetramers, reversible Streptamers, or dye-conjugated pMHC multimers are distinct pMHC reagents tailored for T cell identification, traceless T cell isolation, or TCR characterization, respectively. The specific applicability of each pMHC-based reagent is made possible either through conjugation of probes or reversible multimerization in separate production processes, which is laborious, time-consuming, and prone to variability between the different types of pMHC reagents. This prohibits broad implementation of different types of pMHC reagents as a standard toolbox in routine clinical immune monitoring and immunotherapy. In this article, we describe a novel method for fast and standardized generation of any pMHC multimer reagent from a single precursor (“FLEXamer”). FLEXamers unite reversible multimerization and versatile probe conjugation through a novel double tag (Strep-tag for reversibility and Tub-tag for versatile probe conjugation). We demonstrate that FLEXamers can substitute conventional pMHC reagents in all state-of-the-art applications, considerably accelerating and standardizing production without sacrificing functional performance. Although FLEXamers significantly aid the applicability of pMHC-based reagents in routine workflows, the double tag also provides a universal tool for the investigation of transient molecular interactions in general. Weniger anzeigen

Nanobodies can be considered as next-generation life science tools for the recognition and modulation of antigens that are inaccessible to conventional antibodies. Due to their compact structure and high stability, nanobodies see frequent usage in basic research. Their chemical functionalization facilitates powerful diagnostic tools and opens the way towards promising therapeutic applications. In this review, central aspects of nanobody functionalization are given together with selected… Mehr anzeigen

Nanobodies can be considered as next-generation life science tools for the recognition and modulation of antigens that are inaccessible to conventional antibodies. Due to their compact structure and high stability, nanobodies see frequent usage in basic research. Their chemical functionalization facilitates powerful diagnostic tools and opens the way towards promising therapeutic applications. In this review, central aspects of nanobody functionalization are given together with selected applications in molecular cell biology. While first-generation conjugation strategies rely on random modification of natural amino acids, more recent studies focus on a site-specific attachment of functional groups. Such techniques include chemoenzymatic approaches, expressed protein ligation and amber suppression in combination with bioorthogonal modification strategies. With an ever growing toolkit of protein synthesis and conjugation, functional applications are on the rise as well. Such recent applications range from sophisticated imaging and mass spectrometry to the delivery of nanobodies into living cells, enabling visualization and manipulation of intracellular antigens. Weniger anzeigen

Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich… Mehr anzeigen

Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein–protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets. Weniger anzeigen

The broad substrate tolerance of tubulin tyrosine ligase is the basic rationale behind its wide applicability for chemoenzymatic protein functionalization. In this context, we report that the wild-type enzyme enables ligation of various unnatural amino acids that are substantially bigger than and structurally unrelated to the natural substrate, tyrosine, without the need for extensive protein engineering. This unusual substrate flexibility is due to the fact that the enzyme's catalytic pocket… Mehr anzeigen

The broad substrate tolerance of tubulin tyrosine ligase is the basic rationale behind its wide applicability for chemoenzymatic protein functionalization. In this context, we report that the wild-type enzyme enables ligation of various unnatural amino acids that are substantially bigger than and structurally unrelated to the natural substrate, tyrosine, without the need for extensive protein engineering. This unusual substrate flexibility is due to the fact that the enzyme's catalytic pocket forms an extended cavity during ligation, as confirmed by docking experiments and all-atom molecular dynamics simulations. This feature enabled one-step C-terminal biotinylation and fluorescent coumarin labeling of various functional proteins as demonstrated with ubiquitin, an antigen binding nanobody, and the apoptosis marker Annexin V. Its broad substrate tolerance establishes tubulin tyrosine ligase as a powerful tool for in vitro enzyme-mediated protein modification with single functional amino acids in a specific structural context. Weniger anzeigen

Antibody drug conjugates (ADCs), a promising class of cancer biopharmaceuticals, combine the specificity of therapeutic antibodies with the pharmacological potency of chemical, cytotoxic drugs. Ever since the first ADCs on the market, a plethora of novel ADC technologies has emerged, covering as diverse aspects as antibody engineering, chemical linker optimization and novel conjugation strategies, together aiming at constantly widening the therapeutic window for ADCs. This review primarily… Mehr anzeigen

Antibody drug conjugates (ADCs), a promising class of cancer biopharmaceuticals, combine the specificity of therapeutic antibodies with the pharmacological potency of chemical, cytotoxic drugs. Ever since the first ADCs on the market, a plethora of novel ADC technologies has emerged, covering as diverse aspects as antibody engineering, chemical linker optimization and novel conjugation strategies, together aiming at constantly widening the therapeutic window for ADCs. This review primarily focuses on novel chemical and biotechnological strategies for the site-directed attachment of drugs that are currently validated for 2nd generation ADCs to promote conjugate homogeneity and overall stability. Weniger anzeigen

A novel chemoenzymatic approach for simple and fast site-specific protein labeling is reported. Recombinant tubulin tyrosine ligase (TTL) was repurposed to attach various unnatural tyrosine derivatives as small bioorthogonal handles to proteins containing a short tubulin-derived recognition sequence (Tub-tag). This novel strategy enables a broad range of high-yielding and fast chemoselective C-terminal protein modifications on isolated proteins or in cell lysates for applications in… Mehr anzeigen

A novel chemoenzymatic approach for simple and fast site-specific protein labeling is reported. Recombinant tubulin tyrosine ligase (TTL) was repurposed to attach various unnatural tyrosine derivatives as small bioorthogonal handles to proteins containing a short tubulin-derived recognition sequence (Tub-tag). This novel strategy enables a broad range of high-yielding and fast chemoselective C-terminal protein modifications on isolated proteins or in cell lysates for applications in biochemistry, cell biology, and beyond, as demonstrated by the site-specific labeling of nanobodies, GFP, and ubiquitin. Weniger anzeigen

The total synthesis of solandelactones A and B is presented. The eastern cyclopropyl moiety was prepared following an exceptionally short chemoenzymatic approach whereas enantioselective synthesis of the western side-chain relied on the application of diastereomerically pure allyl boronates. The natural products solandelactones A and B were isolated in good overall yields following convergence of each eastern and western element by application of the Nozaki–Hiyama–Kishi reaction.

The quest to enlarge the molecular space of functional biomolecules has led to the discovery of selective, mild and high-yielding chemical reactions for the modification of peptides and proteins. These conjugation methods have recently become even more advanced with the advent of modern biochemical techniques such as unnatural protein expression or enzymatic reactions that allow the site-specific modification of proteins.

Within this overview, we will highlight recent examples that… Mehr anzeigen

The quest to enlarge the molecular space of functional biomolecules has led to the discovery of selective, mild and high-yielding chemical reactions for the modification of peptides and proteins. These conjugation methods have recently become even more advanced with the advent of modern biochemical techniques such as unnatural protein expression or enzymatic reactions that allow the site-specific modification of proteins.

Within this overview, we will highlight recent examples that describe the site-specific functionalization of proteins. These examples go beyond the straightforward attachment of a given functional moiety to the protein backbone by employing either an innovative linker-design or by novel conjugation chemistry, where the modification reaction itself is responsible for the (altered) functional behaviour of the biomolecule. The examples covered herein include ‘turn-on’ probes for cellular imaging with low levels of background fluorescence, branched or cleavable polymer–protein conjugates of high stability within a cellular environment, the installation of natural occurring posttranslational modifications to help understand their role in complex cellular environments and finally the engineering of novel antibody drug conjugates to facilitate target specific drug release. Weniger anzeigen