This overview guides both novices and experienced researchers facing challenging targets to select the most appropriate gene expression system for producing a particular protein. By answering four key questions, readers can determine the most suitable gene expression system following a decision scheme. This guide addresses the most commonly used and accessible systems and provides brief descriptions of the main gene expression systems' key characteristics to assist decision making. Additionally, information has been included for selected less frequently used "exotic" gene expression systems.

The NF-κB essential modulator NEMO is the core regulatory component of the inhibitor of κB kinase complex, which is a critical checkpoint in canonical NF-κB signaling downstream of innate and adaptive immune receptors. In response to various stimuli, such as TNF or IL-1β, NEMO binds to linear or M1-linked ubiquitin chains generated by LUBAC, promoting its oligomerization and subsequent activation of the associated kinases. Here we show that M1-ubiquitin chains induce phase separation of NEMO and the formation of NEMO assemblies in cells after exposure to IL-1β. Phase separation is promoted by both binding of NEMO to linear ubiquitin chains and covalent linkage of M1-ubiquitin to NEMO and is essential but not sufficient for its phase separation. Supporting the functional relevance of NEMO phase separation in signaling, a pathogenic NEMO mutant, which is impaired in both binding and linkage to linear ubiquitin chains, does not undergo phase separation and is defective in mediating IL-1β–induced NF-κB activation.

Misregulation and mutations of the transcription factor Nrf2 are involved in the development of a variety of human diseases. In this study, we employed the technology of stapled peptides to address a protein-DNA-complex and designed a set of Nrf2-based derivatives. Varying the length and position of the hydrocarbon staple, we chose the best peptide for further evaluation in both fixed and living cells. Peptide 4 revealed significant enrichment within the nucleus compared to its linear counterpart 5, indicating potent binding to DNA. Our studies suggest that these molecules offer an interesting strategy to target activated Nrf2 in cancer cells.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

The delivery of small molecule fluorophores with minimal compartmentalization is currently one of the most critical technical problems in intracellular labelling. Here we introduce sulfonated and phosphonated coumarin dyes, demonstrate rapid cell entry via a prodrug approach, and show a lack of interaction with membranes, organelles, or other compartments. The dyes show no specific localization and are evenly distributed in the cells. Our fluorogenic, clickable phosphonate derivatives successfully tagged model targets in intact cells and the increase in brightness upon click reaction was around 60-fold.

Identification and analysis of small molecule bioactivity in target-agnostic cellular assays and monitoring changes in phenotype followed by identification of the biological target are a powerful approach for the identification of novel bioactive chemical matter in particular when the monitored phenotype is disease-related and physiologically relevant. Profiling methods that enable the unbiased analysis of compound-perturbed states can suggest mechanisms of action or even targets for bioactive small molecules and may yield novel insights into biology. Here we report the enantioselective synthesis of natural-product-inspired 8-oxotetrahydroprotoberberines and the identification of Picoberin, a low picomolar inhibitor of Hedgehog (Hh)-induced osteoblast differentiation. Global transcriptome and proteome profiling revealed the aryl hydrocarbon receptor (AhR) as the molecular target of this compound and identified a cross talk between Hh and AhR signaling during osteoblast differentiation.

Neutralizing antibodies against SARS-CoV-2 are important to protect against infection and/or disease. Using an assay to detect antibodies directed against the receptor binding domain (RBD) of SARS-CoV-2 Spike, we identified individuals with SARS-CoV-2 infection after an outbreak at a local health institution. All but one COVID-19 patient developed detectable anti-RBD antibodies and 77% had virus neutralizing antibody titers of >1:25. Antibody levels declined slightly over time. However, we still detected virus neutralizing antibody titers in 64% of the COVID-19 patients at >300 days after infection, demonstrating durability of neutralizing antibody levels after infection. Importantly, full COVID-19 vaccination of these individuals resulted in higher antibody titers compared to fully vaccinated individuals in the absence of prior infection. These data demonstrate long-lived antibody-mediated immunity after SARS-CoV-2 infection, and a clear benefit of two vaccine doses for recovered individuals.

Genetic code expansion is a powerful tool for the study of protein interactions, as it allows for the site-specific incorporation of a photoreactive group via non-canonical amino acids. Recently, several groups have published bifunctional amino acids that carry a handle for click chemistry in addition to the photo-crosslinker. This allows for the specific labeling of crosslinked proteins and therefore the pulldown of peptides for further analysis. This review describes the properties and advantages of different bifunctional amino acids, and gives an overview about current and future applications.

Ultraviolent crosslinking is a key experimental step in the numerous protocols that have been developed for capturing and dissecting RNA-protein interactions in living cells. UV crosslinking covalently stalls dynamic interactions between RNAs and the directly contacting RNA-binding proteins and enables stringent denaturing downstream purification conditions needed for the enrichment and biochemical analysis of RNA-protein complexes. Despite its popularity, conventional 254 nm UV crosslinking possesses a set of intrinsic drawbacks, with the low photochemical efficiency being the central caveat. Here we show that genetically encoded photoreactive unnatural amino acids bearing a dialkyl diazirine photoreactive group can address this problem. Using the human iron regulatory protein 1 (IRP1) as a model RNA-binding protein, we show that the photoreactive amino acids can be introduced into the protein without diminishing its RNA-binding properties. A sevenfold increase in the crosslinking efficiency compared to conventional 254 nm UV crosslinking was achieved using the diazirine-based unnatural amino acid DiAzKs. This finding opens an avenue for new applications of the unnatural amino acids in studying RNA-protein interactions.

Mapping of weak and hence transient interactions between low-abundance interacting molecules is still a major challenge in systems biology and protein biochemistry. Therefore, additional system-wide acting tools are needed to determine protein interactomics. Most important are reagents that can be applied at any kind of protein interface and the possibility to enrich cross-linked fragments with high efficiency. In this study, we report the synthesis of a novel noncanonical amino acid that features a diazirine group for ultraviolet cross-linking as well as an alkyne group for labeling by click chemistry. This bifunctional amino acid, called PrDiAzK, may be inserted into almost any protein interface with minimal structural perturbation using genetic code expansion. We demonstrate that PrDiAzK can be site-selectively incorporated into proteins in both bacterial and mammalian cell cultures, and we show that PrDiAzK allows protein labeling as well as cross-linking. In addition, we tested PrDiAzK for proteome-wide incorporation via stochastic orthogonal recoding of translation, implying potential applications in system-wide mapping of protein-protein interactions in the future.

A method for producing multiprotein complexes engineered with site–specifically introduced noncanonical amino acids is described, enabling applications in biochemical and biophysical analysis, as well as in biotechnology. We present a baculovirus-based protein engineering method that enables site-specific introduction of unique functionalities in a eukaryotic protein complex recombinantly produced in insect cells. We demonstrate the versatility of this efficient and robust protein production platform, 'MultiBacTAG', (i) for the fluorescent labeling of target proteins and biologics using click chemistries, (ii) for glycoengineering of antibodies, and (iii) for structure–function studies of novel eukaryotic complexes using single-molecule Förster resonance energy transfer as well as site-specific crosslinking strategies.

The regulation of many protein kinases by binding to calcium/calmodulin connects two principal mechanisms in signaling processes: protein phosphorylation and responses to dose-and time-dependent calcium signals. We used the calcium/calmodulin-dependent members of the death-associated protein kinase (DAPK) family to investigate the role of a basic DAPK signature loop near the kinase active site. In DAPK2, this loop comprises a novel dimerization-regulated calcium/calmodulin-binding site, in addition to a well-established calcium/calmodulin site in the C-terminal autoregulatory domain. Unexpectedly, impairment of the basic loop interaction site completely abolishes calcium/calmodulin binding and DAPK2 activity is reduced to a residual level, indicative of coupled binding to the two sites. This contrasts with the generally accepted view that kinase calcium/calmodulin interactions are autonomous of the kinase catalytic domain. Our data establish an intricate model of multi-step kinase activation and expand our understanding of how calcium binding connects with other mechanisms involved in kinase activity regulation.

trans-Cyclooctene groups incorporated into proteins via non-canonical amino acids (ncAAs) are emerging as specific handles for bioorthogonal chemistry. Here, we present a highly improved synthetic access to the axially and the equatorially linked trans-cyclooct-2-ene isomers (1 a,b). We further show that the axially connected isomer has a half-life about 10 times higher than the equatorial isomer and reacts with tetrazines much faster, as determined by stopped-flow experiments. The improved properties resulted in different labeling performance of the insulin receptor on the surface of intact cells.

Copper-free click chemistry is currently the most promising and most rapidly developing technology for performing tailored chemical reactions inside intact living cells and animals. Its potential is particularly intensely explored in the field of live cell imaging, for both proteins and metabolites. Here we expand the application spectrum of click reactions to the chemical crosslinking of two proteins of choice in living cells. By combining strain-promoted Diels-Alder cycloaddition with FlAsH-based labeling of peptidic tetracysteine motifs, we developed the membrane-permeating reversible crosslinker T-CrAsH. We demonstrate the feasibility of the method both in vitro and inside cells. The biggest advantage of this new tool is the small size of the crosslinkable groups; this significantly decreases the risk of functional interference.

Chemically induced dimerization (CID) has proven to be a powerful tool for modulating protein interactions. However, the traditional dimerizer rapamycin has limitations in certain in vivo applications because of its slow reversibility and its affinity for endogenous proteins. Described herein is a bioorthogonal system for rapidly reversible CID. A novel dimerizer with synthetic ligand of FKBP' (SLF') linked to trimethoprim (TMP). The SLF' moiety binds to the F36V mutant of FK506-binding protein (FKBP) and the TMP moiety binds to E. coli dihydrofolate reductase (eDHFR). SLF'-TMP-induced heterodimerization of FKBP(F36V) and eDHFR with a dissociation constant of 0.12 μM. Addition of TMP alone was sufficient to rapidly disrupt this heterodimerization. Two examples are presented to demonstrate that this system is an invaluable tool, which can be widely used to rapidly and reversibly control protein function in vivo.

How can the integrin adhesome get self-assembled locally, rapidly, and correctly as diverse cell-matrix adhesion sites? Here, we investigate this question by exploring the cytosolic state of integrin-adhesome components and their dynamic exchange between adhesion sites and cytosol. Using fluorescence cross-correlation spectroscopy (FCCS) and fluorescence recovery after photobleaching (FRAP) we found that the integrin adhesome is extensively pre-assembled already in the cytosol as multi-protein building blocks for adhesion sites. Stationary focal adhesions release symmetrically the same types of protein complexes that they recruit, thereby keeping the cytosolic pool of building blocks spatiotemporally uniform. We conclude a model in which multi-protein building blocks enable rapid and modular self-assembly of adhesion sites and symmetric exchange of these building blocks preserves their specifications and thus the assembly logic of the system.