RESUMEN
Many promising therapeutic protein or peptide drug candidates are rapidly excreted from an organism due to their small size or their inherent immunogenicity. One way to counteract these effects is PEGylation, in which the biopolymer is shielded by synthetic polymers exploiting their stealth properties. However, these modifications are often accompanied by a reduction in the biological function of the protein. By using responsive moieties that bridge the polymer to the protein, a reversible character is provided to this type of conjugation. In this regard, the reductive-responsive nature of disulfides can be exploited via self-immolative structures for reversible linkage to aminic lysine residues and the N-terminus on the protein surface. They enable a traceless release of the intact protein without any further modification and thus preserve the protein's bioactivity. In this study, we demonstrate how this chemistry can be made broadly accessible to RAFT-derived water-soluble polymers like poly(N,N-dimethylacrylamide) (pDMA) as a relevant PEG alternative. A terminal reactive imidazole carbamate with an adjacent self-immolative motif was generated in a gradual manner onto the trithiocarbonate chain transfer moiety of the polymer by first substituting it with a disulfide-bridged alcohol and subsequently converting it into an amine reactive imidazole carbamate. Successful synthesis and complete characterization were demonstrated by NMR, size exclusion chromatography, and mass spectrometry. Finally, two model proteins, lysozyme and a therapeutically relevant nanobody, were functionalized with the generated polymer, which was found to be fully reversible under reductive conditions in the presence of free thiols. This strategy has the potential to extend the generation of reversible reductive-responsive polymer-protein hybrids to the broad field of available functional RAFT-derived polymers.
Asunto(s)
Polímeros , Proteínas , Proteínas/química , Aminas/química , Carbamatos , ImidazolesRESUMEN
The desire to create biomolecules modified with functionalities that go beyond nature's toolbox has resulted in the development of biocompatible and selective methodologies and reagents, each with different scope and limitations. In this overview, we highlight recent advances in the field of bioconjugation from 2016 to 2023. First, (metal-mediated) protein functionalization by exploiting the specific reactivity of amino acids will be discussed, followed by novel bioorthogonal reagents for bioconjugation of modified biomolecules.
Asunto(s)
Aminoácidos , BioingenieríaRESUMEN
Multimeric and polymeric proteins are large biomacromolecules consisting of multiple protein molecules as their monomeric units, connected through covalent or non-covalent bonds. Genetic modification and post-translational modifications (PTMs) of proteins offer alternative strategies for designing and creating multimeric and polymeric proteins. Multimeric proteins are commonly prepared by genetic modification, whereas polymeric proteins are usually created through PTMs. There are two methods that can be applied to create polymeric proteins: self-assembly and crosslinking. Self-assembly offers a spontaneous reaction without a catalyst, while the crosslinking reaction offers some catalyst options, such as chemicals and enzymes. In addition, enzymes are excellent catalysts because they provide site-specificity, rapid reaction, mild reaction conditions, and activity and functionality maintenance of protein polymers. However, only a few enzymes are applicable for the preparation of protein polymers. Most of the other enzymes are effective only for protein conjugation or labeling. Here, we review novel and applicable strategies for the preparation of multimeric proteins through genetic modification and self-assembly. We then describe the formation of protein polymers through site-selective crosslinking reactions catalyzed by enzymes, crosslinking reactions of non-natural amino acids, and protein-peptide (SpyCatcher/SpyTag) interactions. Finally, we discuss the potential applications of these protein polymers.
Asunto(s)
Polímeros , Proteínas , Catálisis , Polimerizacion , Multimerización de Proteína , Proteínas/químicaRESUMEN
A biomolecule-guided self-assembly is a powerful approach to systematically organize diverse inorganic nanoparticles into predefined nanostructures in multiple dimensions. A class of supramolecular proteins is one kind of such biomolecules natively possessing exquisite structures and modifiable ligands, providing a desired candidate for templating functional nanoparticles. Indeed, protein-based assembly of nano-objects has been emerging as one of the ideal routes to fabricate precise architectures. Here, we briefly summarize recent works of well-organized nanoparticle structures templated by individual proteins or highly ordered protein assemblies. The functionalization of protein templates and control over the interactions between nanoparticles and templates are highlighted. Finally, current challenges and future directions are discussed in the design of complicated protein-based materials.
Asunto(s)
Materiales Biocompatibles/química , Nanoestructuras/química , Proteínas/química , Ensayo de Materiales , Tamaño de la PartículaRESUMEN
Stem cell-derived, organotypic in vitro models, known as organoids, have emerged as superior alternatives to traditional cell culture models due to their unparalleled ability to recreate complex physiological and pathophysiological processes. For this reason, they are attractive targets of tissue-engineering efforts, as constructs that include organoid technology would be expected to better simulate the many functions of the desired tissue or organ. While the 3D spheroidal architecture that is the default architecture of most organoid models may be preferred for some applications, 2D monolayer arrangements remain the preferred organization for many applications in tissue engineering. Therefore, in this work, we present a method to create monolayer organoid cultures on poly(ethylene glycol) (PEG) hydrogel scaffolds, using intestinal epithelial organoids (IEOs) as a proof-of-concept. Our process involves two steps: the hydrogel is first functionalized with a layer of poly(D-lysine) (PDL), which then allows the adsorption of pristine, unmodified basement membrane proteins. This approach successfully mediates the formation of IEO monolayer unlike conventional approaches that rely on covalent modification of the hydrogel surface with cell-adhesive peptides and basement membrane proteins. We show that these IEO monolayers recreate important physiological functions of the native intestinal epithelium, including multilineage differentiation, apical-basal polarization, and the ability to model infections with human norovirus. We also show coating of a scaffold mimicking intestinal villous topography, resulting in a 3D IEO monolayer. We expect that this protocol will be useful to researchers attempting to leverage the increased physiological relevance of organoid models to elevate the potential of their tissue-engineered constructs. Impact statement While organoids are physiologically superior models of biological functions than traditional cell cultures, their 3D spheroidal architecture is an obstacle to their incorporation in many tissue-engineering applications, which often prefer 2D monolayer arrangements of cells. For this reason, we developed a protocol to establish monolayer cultures of organoids on poly(ethylene glycol) hydrogels and demonstrate its utility using intestinal epithelial organoids as a proof-of-concept. We expect that this protocol will be of use to researchers creating engineered tissues for both regenerative medicine applications, as well as advanced in vitro experimental models.
Asunto(s)
Hidrogeles , Organoides , Materiales Biocompatibles , Técnicas de Cultivo de Célula , Humanos , PolietilenglicolesRESUMEN
Three-dimensional (3D) control over the placement of bioactive cues is fundamental to understand cell guidance and develop engineered tissues. Two-photon patterning (2PP) provides such placement at micro- to millimeter scale, but nonspecific interactions between proteins and functionalized extracellular matrices (ECMs) restrict its use. Here, a 2PP system based on nonfouling hydrophilic photocages and Sortase A (SA)-based enzymatic coupling is presented, which offers unprecedented orthogonality and signal-to-noise ratio in both inert hydrogels and complex mammalian matrices. Improved photocaged peptide synthesis and protein functionalization protocols with broad applicability are introduced. Importantly, the method enables 2PP in a single step in the presence of fragile biomolecules and cells, and is compatible with time-controlled growth factor presentation. As a corollary, the guidance of axons through 3D-patterned nerve growth factor (NGF) within brain-mimetic ECMs is demonstrated. The approach allows for the interrogation of the role of complex signaling molecules in 3D matrices, thus helping to better understand biological guidance in tissue development and regeneration.
Asunto(s)
Matriz Extracelular/química , Factor de Crecimiento Nervioso/química , Aminoaciltransferasas/química , Aminoaciltransferasas/metabolismo , Animales , Axones/química , Axones/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cumarinas/química , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Matriz Extracelular/metabolismo , Ácido Hialurónico/química , Hidrogeles/química , Microscopía de Fluorescencia por Excitación Multifotónica , Factor de Crecimiento Nervioso/metabolismo , FotonesRESUMEN
An expression strategy is presented in order to produce nanobodies modified with a clickable alkyne functionality at their C-terminus via the intein-mediated protein ligation (IPL) technique. The protocol focuses on the cytoplasmic expression and extraction of a nanobody-intein-chitin binding domain (CBD) fusion protein in E. coli SHuffle® T7 cells, in the commonly used Luria-Bertani (LB) medium. The combination of these factors results in a high yield and nearly complete alkynation of the nanobody at its C-terminus via IPL. The resulting alkynated nanobodies retain excellent binding capacity toward the nanobody targeted antigen. The presented protocol benefits from time- and cost-effectiveness and allows for a feasible upscaling of functionalized (here alkynated) nanobodies. The production of high quantities of site-specifically modified nanobodies paves the way to (1) novel biosurface applications that demand for homogeneously oriented nanobodies having their active site fully accessible for target (e.g., biomarker) binding, and (2) innovative applications such as localized drug delivery and image guided surgery by covalent "click" chemistry coupling of these alkynated nanobodies to a multitude of azide-containing counterparts as there are drug containing polymers and contrast labeling agents.
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Química Clic/métodos , Inteínas/genética , Ingeniería de Proteínas/métodos , Anticuerpos de Dominio Único/química , Quitina/química , Quitina/genética , Unión Proteica/genética , Dominios Proteicos/genética , Anticuerpos de Dominio Único/genéticaRESUMEN
Tub-tag labeling, a novel chemoenzymatic protein functionalization method, facilitates one-step fluorescent labeling of functional biomolecules. The enzyme tubulin tyrosine ligase incorporates coumarin-amino acids to the terminal carboxylic acid of proteins containing a short peptidic recognition sequence called Tub-tag. Here we describe the one-step Tub-tag protein modification protocol in detail and explain its utilization to generate fluorescently labeled proteins for advanced applications in imaging and diagnostics.
Asunto(s)
Péptido Sintasas/química , Péptidos/química , Proteínas/aislamiento & purificación , Coloración y Etiquetado/métodos , Aminoácidos/química , Colorantes Fluorescentes/química , Proteínas/químicaRESUMEN
Proteins can be labeled site-specifically and in inducible fashion by exposing a small peptide tag (G4Y) on any of its termini and activating the newly exposed tyrosine residue with the enzyme mushroom tyrosinase. The enzyme generates a quinone by oxidizing the tyrosine, which in turn can perform strain-promoted oxidation-controlled ortho-quinone cycloaddition (SPOCQ) with strained alkynes and alkenes, generating a stable conjugation product. Here, we describe a protocol to perform SPOCQ reaction on proteins, along with notes to optimize yield and reaction rates. Conjugation efficiencies of over 95% to antibodies have been reported using this protocol.
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Oxidación-Reducción , Proteínas/química , Coloración y Etiquetado , Tirosina/química , Anticuerpos/química , Catálisis , Cromatografía Líquida de Alta Presión , Humanos , Inmunoconjugados/química , Espectrometría de Masas , Coloración y Etiquetado/métodosRESUMEN
Tubulin tyrosine ligase (TTL) catalyzes the addition of tyrosine derivatives to the C-terminal carboxylic acid of proteins. The enzyme binds to a 14-amino acid recognition sequence, termed Tub-tag, and allows for the introduction of tyrosine derivatives that carry a unique chemical handle. These handles enable subsequent bioorthogonal reactions with a great variety of probes or effector molecules. Clearly, this two-step chemoenzymatic approach, facilitates the site-specific functionalization of proteins. Furthermore, due to its broad substrate tolerance, tubulin tyrosine ligase also enables an enzymatic one-step modification. For example, a coumarin amino acid was utilized to generate fluorescently labeled proteins for advanced applications in imaging and diagnostics. Here we describe the modification of proteins using TTL in detail via a one-step as well as two-step procedure and highlight its practicability for applications in imaging, diagnostics, and cell biology.
Asunto(s)
Péptido Sintasas/metabolismo , Proteínas/metabolismo , Aminoácidos/química , Catálisis , Línea Celular , Humanos , Péptido Sintasas/química , Procesamiento Proteico-Postraduccional , Proteínas/química , Proteínas Recombinantes , Análisis Espectral , Relación Estructura-ActividadRESUMEN
The degradation of damaged proteins is essential for cell viability. Lon is a highly conserved ATP-dependent serine-lysine protease that maintains proteostasis. We performed a comparative genome-wide analysis to determine the evolutionary history of Lon proteases. Prokaryotes and unicellular eukaryotes retained a single Lon copy, whereas multicellular eukaryotes acquired a peroxisomal copy, in addition to the mitochondrial gene, to sustain the evolution of higher order organ structures. Land plants developed small Lon gene families. Despite the Lon2 peroxisomal paralog, Lon genes triplicated in the Arabidopsis lineage through sequential evolutionary events including whole-genome and tandem duplications. The retention of Lon1, Lon4, and Lon3 triplicates relied on their differential and even contrasting expression patterns, distinct subcellular targeting mechanisms, and functional divergence. Lon1 seems similar to the pre-duplication ancestral gene unit, whereas the duplication of Lon3 and Lon4 is evolutionarily recent. In the wider context of plant evolution, papaya is the only genome with a single ancestral Lon1-type gene. The evolutionary trend among plants is to acquire Lon copies with ambiguous pre-sequences for dual-targeting to mitochondria and chloroplasts, and a substrate recognition domain that deviates from the ancestral Lon1 type. Lon genes constitute a paradigm of dynamic evolution contributing to understanding the functional fate of gene duplicates.
Asunto(s)
Evolución Molecular , Duplicación de Gen , Proteínas de Plantas/genética , Plantas/genética , Proteasa La/genética , Secuencia de Bases , Filogenia , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Proteasa La/metabolismo , Alineación de SecuenciaRESUMEN
Tub-tag labeling is a chemoenzymatic method that enables the site-specific labeling of proteins. Here, the natural enzyme tubulin tyrosine ligase incorporates noncanonical tyrosine derivatives to the terminal carboxylic acid of proteins containing a 14-amino acid recognition sequence called Tub-tag. The tyrosine derivative carries a unique chemical reporter allowing for a subsequent bioorthogonal modification of proteins with a great variety of probes. Here, we describe the Tub-tag protein modification protocol in detail and explain its utilization to generate labeled proteins for advanced applications in cell biology, imaging, and diagnostics.