GFP split

Yeast-based secretion systems are advantageous for engineering highly interesting enzymes that are not or barely producible in E. coli. The herein-presented production setup facilitates high-throughput screening as no cell lysis is required. All techniques are described in detail, with access to freely available online tools and all vectors have been made available on the non-profit plasmid repository AddGene. We describe the method for UPOs as a model enzyme, showcasing their secretion, detection, and evolution using S...

Green fluorescent proteins (GFPs) are ubiquitous for protein tagging and live-cell imaging. Split-GFPs are widely used to study protein-protein interactions by fusing proteins of interest to split GFP fragments that create a fluorophore upon typically irreversible complementation. Thus, controlled dissociation of the fragments is desirable. Although we have found that split strands can be photodissociated, the quantum efficiency of light-induced photodissociation of split GFPs is low. Traditional protein engineering approaches to increase efficiency, including extensive mutagenesis and screening, have proved difficult to implement...

Human sirtuin isoform 2 (SIRT2) is an NAD+ -dependent enzyme that functions as a lysine deacetylase and defatty-acylase. Here, we report that SIRT2 readily dimerizes in solution and in cells and that dimerization affects its ability to remove different acyl modifications from substrates. Dimerization of recombinant SIRT2 was revealed with analytical size exclusion chromatography and chemical cross-linking. Dimerized SIRT2 dissociates into monomers upon binding long fatty acylated substrates (decanoyl-, dodecanoyl-, and myristoyl-lysine)...

Sensory neurons enable an organism to perceive external stimuli, which is essential for survival. The sensory capacity of a neuron depends on the elaboration of its dendritic arbor and the localization of sensory ion channels to the dendritic membrane. However, it is not well understood when and how ion channels localize to growing sensory dendrites and whether their delivery is coordinated with growth of the dendritic arbor. We investigated the localization of the DEG/ENaC/ASIC ion channel Pickpocket (Ppk) in the peripheral sensory neurons of developing fruit flies...

Binding ligands empower molecular therapeutics and diagnostics. Despite an array of protein scaffolds engineered for binding, the biophysical elements that drive developability and evolvability are not fully understood. In particular, engineering novel function while maintaining biophysical integrity within the context of small, single-domain proteins is challenged by integration of the structural framework and the evolved binding site. Miniproteins present a challenge to our limits of protein engineering capability and provide advantages in physiological targeting, modularity for multi-functional constructs, and unique binding modes...

Animal development involves numerous molecular events, whose spatiotemporal properties largely determine the biological outcomes. Conventional methods for studying gene function lack the necessary spatiotemporal resolution for precise dissection of developmental mechanisms. Optogenetic approaches are powerful alternatives, but most existing tools rely on exogenous designer proteins that produce narrow outputs and cannot be applied to diverse or endogenous proteins. To address this limitation, we developed OptoTrap, a light-inducible protein trapping system that allows manipulation of endogenous proteins tagged with GFP or split GFP...

All vertebrate genomes encode for three large histone H2A variants that have an additional metabolite-binding globular macrodomain module, macroH2A. MacroH2A variants impact heterochromatin organization and transcription regulation and establish a barrier for cellular reprogramming. However, the mechanisms of how macroH2A is incorporated into chromatin and the identity of any chaperones required for histone deposition remain elusive. Here, we develop a split-GFP-based assay for chromatin incorporation and use it to conduct a genome-wide mutagenesis screen in haploid human cells to identify proteins that regulate macroH2A dynamics...

In the case of the Japanese encephalitis virus (JEV), the envelope protein (E), a major component of viral particles, contains a highly conserved N-linked glycosylation site (E: N154). Glycosylation of the E protein is thought to play an important role in the ability of the virus to attach to target cells during transmission; however, its role in viral particle formation and release remains poorly understood. In this study, we investigated the role of N-glycosylation of flaviviral structural proteins in viral particle formation and secretion by introducing mutations in viral structural proteins or cellular factors involved in glycoprotein transport and processing...

BACKGROUND: Bacillus subtilis is one of the workhorses in industrial biotechnology and well known for its secretion potential. Efficient secretion of recombinant proteins still requires extensive optimization campaigns and screening with activity-based methods. However, not every protein can be detected by activity-based screening. We therefore developed a combined online monitoring system, consisting of an in vivo split GFP assay for activity-independent target detection and an mCherry-based secretion stress biosensor...

Caffeine is considered as one of the most important bioactive components in the popular plant beverages tea, cacao, and coffee, but as a wide-spread plant secondary metabolite its biosynthetic regulation at transcription level remains largely unclear. Here, we report a novel transcription factor Camellia sinensis Senescnece 40 ( CsS40 ) as a caffeine biosynthesis regulator, which was discovered during screening a yeast expression library constructed from tea leaf cDNAs for activation of tea caffeine synthase ( TCS1 ) promoter...

Adenoviruses (AdVs) are being developed for oncolytic or vaccination therapy against existing and emerging conditions. Well-characterized replication-competent human and human primate AdVs expressing multiple payloads are desirable, but their replication in rodent models is limited. To score the timing of adenoviral gene expression in cell cultures, we developed fully replication-competent transcriptional reporter viruses for HAdV-C5, -B3 and -B35. The picornavirus-derived 2A sequence, which induces cotranslational peptide splitting and reinitiation (skipping), was linked to GFP and the fused sequence was inserted C-terminal of the early gene E1A, the intermediate early gene protein IX and the late fiber gene...

The 14-3-3 protein family is a highly conservative member of the acid protein family and plays an important role in regulating a series of important biological activities and various signal transduction pathways. The role of 14-3-3 proteins in regulating starch accumulation still remains largely unknown. To investigate the properties of 14-3-3 proteins, the structures and functions involved in starch accumulation in storage roots were analyzed, and consequently, 16 Me14-3-3 genes were identified. Phylogenetic analysis revealed that Me14-3-3 family proteins are split into two groups (ε and non-ε)...

Determination of per- and polyfluoroalkyl substances (PFAS) in drinking water at the low levels set by regulatory officials has been a major focus for sensor developing researchers. However, it is becoming more apparent that detection of these contaminants in soils, foods and consumer products is relevant and necessary at part per billion and even part per million levels. Here, a fluorescent biosensor for the rapid detection of PFOA was engineered based on human liver fatty acid binding protein (hLFABP). By conjugating circularly permuted green fluorescent protein (cp...

LINC00116 encodes a microprotein first identified as Mitoregulin (MTLN), where it was reported to localize to the inner membrane of mitochondria to regulate fatty acid oxidation and oxidative phosphorylation. These initial discoveries were followed by reports with differing findings about its molecular functions and submitochondrial localization. To clarify the apparent discrepancies, we constructed multiple orthogonal methods of determining the localization of MTLN, including split GFP-based reporters that enable efficient and reliable topology analyses for microproteins...

Small GTPases are highly regulated proteins that control essential signaling pathways through the activity of their effector proteins. Among the RHOA subfamily, RHOB regulates peculiar functions that could be associated with the control of the endocytic trafficking of signaling proteins. Here, we used an optimized assay based on tripartite split-GFP complementation to localize GTPase-effector complexes with high-resolution. The detection of RHOB interaction with the Rhotekin Rho binding domain (RBD) that specifically recognizes the active GTP-bound GTPase, is performed in vitro by the concomitant addition of recombinant GFP1-9 and a GFP nanobody...

Henipaviruses include the deadly zoonotic Nipah (NiV) and Hendra (HeV) paramyxoviruses, which have caused recurring outbreaks in human populations. A hallmark of henipavirus infection is the induction of cell-cell fusion (syncytia), caused by the expression of the attachment (G) and fusion (F) glycoproteins on the surface of infected cells. The interactions of G and F with each other and with receptors on cellular plasma membranes drive both viral entry and syncytia formation and are thus of great interest...

Filamentous fungi are competitive hosts for the production of drugs, proteins, and chemicals. However, their utility is limited by screening methods and low throughput. In this work, a universal high-throughput system for optimizing protein production in filamentous fungi was described. Droplet microfluidics was used to encapsulate large mutant strain pools in biocompatible core-shell microdroplets designed to avoid mycelial punctures and thus sustain prolonged culture. The self-assembled split GFP was then used to characterize the secretory capacity of the strains and isolate strains with superior production titers according to the fluorescence signals...

HES1 (hairy and enhancer of split-1, effector of the NOTCH pathway) plays a role in oocyte maturation and has been detected so far mainly in somatic follicular cells. In this study, we aimed to investigate whether HES1 is present in both compartments of bovine cumulus oocyte complexes (COCs) and whether in vitro maturation itself has an effect on its distribution. We investigated the abundance of HES1 mRNA and protein in bovine COCs characterized by Brilliant-Cresyl-Blue (BCB) stainability by RT-PCR and immunofluorescence before and after in vitro maturation (IVM)...

For several decades, researchers have been using protein-fragment complementation assay (PCA) approaches for biosensing to study protein-protein interaction for a variety of aims, including viral infection, cellular apoptosis, G protein coupled receptor (GPCR) signaling, drug and substrate screening, and protein aggregation and protein editing by CRISPR/Cas9. As a reporter, NanoLuc (NLuc), a smaller and the brightest engineered luciferase derived from deep-sea shrimp Oplophorus gracilirostris , has been found to have many benefits over other luminescent enzymes in PCA...

Clustered protocadherin (Pcdh), a cell adhesion protein, is involved in the self-recognition and non-self-discrimination of neurons by conferring diversity on the cell surface. Although the roles of Pcdh in neurons have been elucidated, it has been challenging to visualize its adhesion activity in neurons, which is a molecular function of Pcdh. Here, we present fluorescent indicators, named IPADs, which visualize the interaction of protocadherin-α4 isoform (α4). IPADs successfully visualize not only homophilic α4 trans -interactions, but also combinatorial homophilic interactions between cells...