GFP split

The barley powdery mildew fungus, Blumeria hordei (Bh), secretes hundreds of candidate secreted effector proteins (CSEPs) to facilitate pathogen infection and colonization. One of these, CSEP0008, is directly recognized by the barley nucleotide-binding leucine-rich-repeat (NLR) receptor MLA1 and therefore is designated AVRA1 . Here, we show that AVRA1 and the sequence-unrelated Bh effector BEC1016 (CSEP0491) suppress immunity in barley. We used yeast two-hybrid next-generation interaction screens (Y2H-NGIS), followed by binary Y2H and in planta protein-protein interactions studies, and identified a common barley target of AVRA1 and BEC1016, the endoplasmic reticulum (ER)-localized J-domain protein HvERdj3B...

BACKGROUND: Microbial cell surface display technology allows immobilizing proteins on the cell surface by fusing them to anchoring motifs, thereby endowing the cells with diverse functionalities. However, the assessment of successful protein display and the quantification of displayed proteins remain challenging. The green fluorescent protein (GFP) can be split into two non-fluorescent fragments, while they spontaneously assemble and emit fluorescence when brought together through complementation...

Cell-free protein synthesis (CFPS) systems offer a versatile platform for a wide range of applications. However, the traditional methods for detecting proteins synthesized in CFPS, such as radioactive labeling, fluorescent tagging, or electrophoretic separation, may be impractical, due to environmental hazards, high costs, technical complexity, and time consuming procedures. These limitations underscore the need for new approaches that streamline the detection process, facilitating broader application of CFPS...

2'-Fucosyllactose (2'-FL), one of the major human milk oligosaccharides, was produced in several engineered microorganisms. However, the low solubility of α-1,2-fucosyltransferase (α1,2-FucT) often becomes a bottleneck to produce maximum amount of 2'-FL in the microorganisms. To overcome this solubility issue, the following studies were conducted to improve the soluble expression of α1,2-FucT. Initially, hydrophobic amino acids in the hydrophilic region of the 6 α-helices were mutated, adhering to the α-helix rule...

Split systems, modular entities enabling controlled biological processes, have become instrumental in biological research. This review highlights their utility across applications like gene regulation, protein interaction identification, and biosensor development. Covering significant progress over the last decade, it revisits traditional split proteins such as GFP, luciferase, and inteins, and explores advancements in technologies like Cas proteins and base editors. We also examine reassembly modules and their applications in diverse fields, from gene regulation to therapeutic innovation...

Mitochondria-ER contact sites (MERCS) are involved in energy homeostasis, redox and Ca2+ signaling, and inflammation. MERCS are heavily studied; however, little is known about their regulation during mitosis. Here, we show that MERCS expand during mitosis in three cell types using various approaches, including transmission electron microscopy, serial EM coupled to 3D reconstruction, and a split GFP MERCS marker. We further show enhanced Ca2+ transfer between the ER and mitochondria using either direct Ca2+ measurements or by quantifying the activity of Ca2+ -dependent mitochondrial dehydrogenases...

Host-pathogen interactions play a critical role in infectious diseases, and understanding the underlying mechanisms is vital for developing effective therapeutic strategies. The visualization and characterization of bacterial proteins within host cells is key to unraveling the dynamics of these interactions. Various protein labeling strategies have emerged as powerful tools for studying host-pathogen interactions, enabling the tracking, localization, and functional analysis of bacterial proteins in real-time...

Mitochondria-ER contact sites (MERCS) serve as hotspots for important cellular processes, including calcium homeostasis, phospholipid homeostasis, mitochondria dynamics, and mitochondrial quality control. MERCS reporters based on complementation of GFP fragments have been designed to visualize MERCS in real-time, but we find that they do not accurately respond to changes in MERCS content. Here, we utilize split LacZ complementing fragments to develop the first MERCS reporter system (termed SpLacZ-MERCS) that continuously integrates the MERCS information within a cell and generates a fluorescent output...

Rationale: Tripeptidyl peptidase II (TPP2) has been proven to be related to human immune and neurological diseases. It is generally considered as a cytosolic protein which forms the largest known protease complex in eukaryotic cells to operate mostly downstream of proteasomes for degradation of longer peptides. However, this canonical function of TPP2 cannot explain its role in a wide variety of biological and pathogenic processes. The mechanistic interrelationships and hierarchical order of these processes have yet to be clarified...

The aim of this study was to evaluate the push-out bond strength and the failure mode of single adjustable posts (SPLENDOR SAP), anatomical posts (AP), or computer-aided design/computer-aided manufacturing (CAD-CAM) posts cemented to flared root canal dentin with self-adhesive dual-cure resin cement. Thirty single-rooted premolars received endodontic treatment with gutta-percha and a calcium hydroxide-based endodontic cement. After 24 hours, the canal spaces were enlarged with burs. The tooth specimens were divided equally into three groups (n=10), according to the glass fiber post (GFP) system: AP-prefabricated GFP (Reforpost #2, Angelus) was relined with composite resin (Filtek Z350 XT, 3M ESPE); SPLENDOR SAP-GFP was used with a glass fiber sleeve to adjust the adaptation (Splendor SAP, Angelus); and CAD-CAM-a resin pattern was obtained and scanned to produce a CAD-CAM GFP (Fiber CAD Lab, Angelus) milled using a subtractive technique...

Pest feeding affects the rhizobacteria community. The rhizomicrobiota activates salicylic acid and jasmonic acid signaling pathways to help plants deal with pest infestation. However, whether plants can recruit special pesticidal microorganisms to deal with attack from herbivores is unclear. A system composed of peanuts and first-instar larvae of Holotrichia parallela were used to analyze whether peanuts truly enrich the insecticidal bacteria after feeding by larvae, and whether inoculation of the enriched bacteria promotes the resistance of plants to herbivore...

Peroxisomes are membrane-bounded organelles that contain enzymes involved in multiple lipid metabolic pathways. Several of these pathways require (re-)activation of fatty acids to coenzyme A (CoA) esters by acyl-CoA synthetases, which may take place inside the peroxisomal lumen or extraperoxisomal. The acyl-CoA synthetases SLC27A2, SLC27A4, ACSL1, and ACSL4 have different but overlapping substrate specificities and were previously reported to be localized in the peroxisomal membrane in addition to other subcellular locations...

The split-GFP system is a versatile tool with numerous applications, but it has been underutilized for the labeling of heterologous surface-displayed proteins. By inserting the 16 amino acid sequence of the GFP11-tag between a protein of interest and an autotransporter protein, it is possible to present a protein at the outer membrane of gram-negative bacteria and to fluorescently label it by complementation with externally added GFP1-10. The labeled cells could be clearly discerned from cells without the protein of interest using flow cytometry and the insertion of the GFP11-tag caused no significant alteration of the catalytic activity for the tested model enzyme CsBglA...

Protein activity is generally functionally integrated and spatially restricted to key locations within the cell. Knocksideways experiments allow researchers to rapidly move proteins to alternate or ectopic regions of the cell and assess the resultant cellular response. Briefly, individual proteins to be tested using this approach must be modified with moieties that dimerize under treatment with rapamycin to promote the experimental spatial relocalizations. CRISPR technology enables researchers to engineer modified protein directly in cells while preserving proper protein levels because the engineered protein will be expressed from endogenous promoters...

We engineered a fluorescent fusion protein of C. elegans lamin, by fusing the eleventh beta strand of GFP to the N-terminus of LMN-1 at the endogenous lmn-1 locus. When co-expressed with GFP1-10, GFP11::LMN-1 was observed at the nuclear periphery of a wide variety of somatic cells. Homozygous gfp11::lmn-1 animals had normal numbers of viable embryos. However, the gfp11::lmn-1 animals had a mild swimming defect. While not completely functional, the GFP11::LMN-1 strain is more healthy than other published fluorescent LMN-1 lines, making it a valuable reagent for studying lamins...

Fluorescent proteins are versatile biomarkers that facilitate effective detection and tracking of macromolecules of interest in real time. Engineered fluorescent proteins such as superfolder green fluorescent protein (sfGFP) and superfolder Cherry (sfCherry) have exceptional refolding capability capable of delivering fluorescent readout in harsh environments where most proteins lose their native functions. Our recent work on the development of a split fluorescent protein from a species of strawberry anemone, Corynactis californica, delivered pairs of fragments with up to three-fold faster complementation than split GFP...

The search for inhibitors of the Ubiquitin Proteasome System (UPS) is an expanding area, due to the crucial role of UPS enzymes in several diseases. The complexity of the UPS and the multiple protein-protein interactions (PPIs) involved, either between UPS proteins themselves or between UPS components and theirs targets, offer an incredibly wide field for the development of chemical compounds for specifically modulating or inhibiting metabolic pathways. However, numerous UPS PPIs are transient/labile, due the processivity of the system (Ubiquitin [Ub] chain elongation, Ub transfer, etc...

Cancer cells inevitably interact with neighboring host tissue-resident cells during the process of metastatic colonization, establishing a metastatic niche to fuel their survival, growth, and invasion. However, the underlying mechanisms in the metastatic niche are yet to be fully elucidated owing to the lack of methodologies for comprehensively studying the mechanisms of cell-cell interactions in the niche. Here, we improve a split green fluorescent protein (GFP)-based genetically encoded system to develop secretory glycosylphosphatidylinositol-anchored reconstitution-activated proteins to highlight intercellular connections (sGRAPHIC) for efficient fluorescent labeling of tissue-resident cells that neighbor on and putatively interact with cancer cells in deep tissues...

Eukaryotic cells use calcium ions (Ca2+) as second messengers, particularly in response to abiotic and biotic stresses. These signals are detected by Ca2+ sensor proteins, such as calmodulin (CaM), which regulate downstream target proteins. Plants also possess many CaM-like proteins (CMLs), most of which remain unstudied. We recently demonstrated that Arabidopsis CML13 and CML14 interact with proteins containing isoleucine/glutamine (IQ) domains, including CaM-binding transcriptional activators (CAMTAs). Here, we show that CaM, CML13, and CML14 bind all six members of the Arabidopsis CAMTA family...

Bacterial micro-compartments (BMC) are complex macromolecular assemblies that participate in varied metabolic processes in about 20% of bacterial species. Most of these organisms carry BMC genetic information organized in operons that often include several paralog genes coding for components of the compartment shell. BMC shell constituents can be classified depending on their oligomerization state as hexamers (BMC-H), pentamers (BMC-P) or trimers (BMC-T). Formation of hetero-oligomers combining different protein homologs is theoretically feasible, something that could ultimately modify BMC shell rigidity or permeability, for instance...