Split Green Fluorescent Protein Research Articles

Creating a bacterium that forms eukaryotic nucleosome core particles

The nucleosome is one of the hallmarks of eukaryotes, a dynamic platform that supports many critical functions in eukaryotic cells. Here, we engineer the in vivo assembly of the nucleosome core in the model bacterium Escherichia coli. We show that bacterial chromosome DNA and eukaryotic histones can assemble in vivo to form nucleosome complexes with many features resembling those found in eukaryotes. The formation of nucleosomes in E. coli was visualized with atomic force microscopy and using tripartite split green fluorescent protein. Under a condition that moderate histones expression was induced at 1 µM IPTG, the nucleosome-forming bacterium is viable and has sustained growth for at least 110 divisions in longer-term growth experiments. It exhibits stable nucleosome formation, a consistent transcriptome across passages, and reduced growth fitness under stress conditions. In particular, the nucleosome arrays in E. coli genic regions have profiles resembling those in eukaryotic cells. The observed compatibility between the eukaryotic nucleosome and the bacterial chromosome machinery may reflect a prerequisite for bacteria-archaea union, providing insight into eukaryogenesis and the origin of the nucleosome.

A High-Throughput Screening Platform for Engineering Poly(ethylene Terephthalate) Hydrolases.

The ability of enzymes to hydrolyze the ubiquitous polyester, poly(ethylene terephthalate) (PET), has enabled the potential for bioindustrial recycling of this waste plastic. To date, many of these PET hydrolases have been engineered for improved catalytic activity and stability, but current screening methods have limitations in screening large libraries, including under high-temperature conditions. Here, we developed a platform that can simultaneously interrogate PET hydrolase libraries of 104-105 variants (per round) for protein solubility, thermostability, and activity via paired, plate-based split green fluorescent protein and model substrate screens. We then applied this platform to improve the performance of a benchmark PET hydrolase, leaf-branch compost cutinase, by directed evolution. Our engineered enzyme exhibited higher catalytic activity relative to the benchmark, LCC-ICCG, on amorphous PET film coupon substrates (∼9.4% crystallinity) in pH-controlled bioreactors at both 65 °C (8.5% higher conversion at 48 h and 38% higher maximum rate, at 2.9% substrate loading) and 68 °C (11.2% higher conversion at 48 h and 43% higher maximum rate, at 16.5% substrate loading), up to 48 h, highlighting the potential of this screening platform to accelerate enzyme development for PET recycling.

Cell-type-specific Labeling of Endogenous Proteins Using the Split GFP System in Drosophila.

i. Accurate identification of the locations of endogenous proteins is crucial for understanding their functions in tissues and cells. However, achieving precise cell-type-specific labeling of proteins has been challenging in vivo . A notable solution to this challenge is the self-complementing split green fluorescent protein (GFP 1-10/11 ) system. In this paper, we present a detailed protocol for labeling endogenous proteins in a cell-type-specific manner using the GFP 1-10/11 system in fruit flies. This approach depends on the reconstitution of the GFP 1-10 and GFP 11 fragments, creating a fluorescence signal. We insert the GFP 11 fragment into a specific genomic locus while expressing its counterpart, GFP 1-10 , through an available Gal4 driver line. The unique aspect of this system is that neither GFP 1-10 nor GFP 11 alone emits fluorescence, enabling the precise detection of protein localization only in Gal4-positive cells expressing the GFP 11 tagged endogenous protein. We illustrate this technique using the adhesion molecule gene teneurin-m ( Ten-m ) as a model, highlighting the generation and validation of GFP 11 protein trap lines via Minos-mediated integration cassette (MiMIC) insertion. Furthermore, we demonstrate the cell-type-specific labeling of Ten-m proteins in the larval brains of fruit flies. This method significantly enhances our ability to image endogenous protein localization patterns in a cell-type-specific manner and is adaptable to various model organisms beyond fruit flies.

Chinese hamster ovary cell line engineering strategies for modular production of custom extracellular vesicles.

Continuously secreted by all cell types, extracellular vesicles (EVs) are small membrane-bound structures which shuttle bioactive cargo between cells across their external environment. Their central role as natural molecular messengers and ability to cross biological barriers has garnered significant attention in the use of EVs as therapeutic delivery vehicles. Still, harnessing the potential of EVs is faced with many obstacles. A cell line engineering approach can be used to exploit EVs to encapsulate a bespoke cargo of interest. However, full details regarding native EV-loading mechanisms remain under debate, making this a challenge. While Chinese hamster ovary (CHO) cells are well known to be the preferred host for recombinant therapeutic protein production, their application as an EV producer cell host has been largely overlooked. In this study, we engineered CHO DG44 cells to produce custom EVs with bespoke cargo. To this end, genetic constructs employing split green fluorescent protein technology were designed for tagging both CD81 and protein cargoes to enable EV loading via self-assembling activity. To demonstrate this, NanoLuc and mCherry were used as model reporter cargoes to validate engineered loading into EVs. Experimental findings indicated that our custom EV approach produced vesicles with up to 15-fold greater cargo compared with commonly used passive loading strategies. When applied to recipient cells, we observed a dose-dependent increase in cargo activity, suggesting successful delivery of engineered cargo via our custom CHO EVs.

Quantitative measurement of cell-surface displayed proteins based on split-GFP assembly

BackgroundMicrobial 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. Based on split-GFP assembly, we aim to: (1) confirm the success display of passenger proteins, (2) quantify the number of passenger proteins displayed on individual cells.ResultsIn this study, we propose two innovative methods based on split-green fluorescent protein (split-GFP), named GFP1-10/GFP11 and GFP1-9/GFP10-11 assembly, for the purpose of confirming successful display and quantifying the number of proteins displayed on individual cells. We evaluated the display efficiency of SUMO and ubiquitin using different anchor proteins to demonstrate the feasibility of the two split-GFP assembly systems. To measure the display efficiency of functional proteins, laccase expression was measured using the split-GFP assembly system by co-displaying GFP11 or GFP10-11 tags, respectively.ConclusionsOur study provides two split-GFP based methods that enable qualitative and quantitative analyses of individual cell display efficiency with a simple workflow, thus facilitating further comprehensive investigations into microbial cell surface display technology. Both split-GFP assembly systems offer a one-step procedure with minimal cost, simplifying the fluorescence analysis of surface-displaying cells.

Sequence-developability mapping of affibody and fibronectin paratopes via library-scale variant characterization.

Protein developability is requisite for use in therapeutic, diagnostic, or industrial applications. Many developability assays are low throughput, which limits their utility to the later stages of protein discovery and evolution. Recent approaches enable experimental or computational assessment of many more variants, yet the breadth of applicability across protein families and developability metrics is uncertain. Here, three library-scale assays-on-yeast protease, split green fluorescent protein (GFP), and non-specific binding-were evaluated for their ability to predict two key developability outcomes (thermal stability and recombinant expression) for the small protein scaffolds affibody and fibronectin. The assays' predictive capabilities were assessed via both linear correlation and machine learning models trained on the library-scale assay data. The on-yeast protease assay is highly predictive of thermal stability for both scaffolds, and the split-GFP assay is informative of affibody thermal stability and expression. The library-scale data was used to map sequence-developability landscapes for affibody and fibronectin binding paratopes, which guides future design of variants and libraries.

Secretory GFP reconstitution labeling of neighboring cells interrogates cell–cell interactions in metastatic niches

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. The sGRAPHIC system enables the isolation of metastatic niche-associated tissue-resident cells for their characterization using a single-cell RNA sequencing platform. We use this sGRAPHIC-leveraged transcriptomic platform to uncover gene expression patterns in metastatic niche-associated hepatocytes in a murine model of liver metastasis. Among the marker genes of metastatic niche-associated hepatocytes, we identify Lgals3, encoding galectin-3, as a potential pro-metastatic factor that accelerates metastatic growth and invasion.

Analyzing Trophoblast Fusion Using Immunofluorescence and Split Protein Complementation Assays.

The fusion of cytotrophoblasts into a multinucleatedsyncytiotrophoblast is essential for placental development. For studies investigating syncytiotrophoblast formation, various methods are available to analyze the fusion efficiency of trophoblast cells in vitro. Here, we describe protocols for measuring trophoblast fusion using immunofluorescence and an assay employing complementary parts of a split green fluorescent protein that self-reassociates and generates a fluorescent signal following cell fusion. Together, these approaches allow for a comprehensive and robust analysis of the fusion index in trophoblast cells and can strengthen the accuracy and throughput of investigations into factors that may regulate syncytiotrophoblast development.

PoreGlow: A split green fluorescent protein-based system for rapid detection of Listeria monocytogenes

Listeria monocytogenes, a severe foodborne pathogen causing severe diseases underscores the necessity for the development of a detection system with high specificity, sensitivity and utility. Herein, the PoreGlow system, based on split green fluorescent protein (GFP), was developed and assessed for the fast and accurate detection of L. monocytogenes. Split GFP-encapsulated liposomes were optimized for targeted analysis. The system utilizes listeriolysin O (LLO), a toxin produced by L. monocytogenes that enlarges the pores split GFP-encapsulated liposomes, to detect L. monocytogenes by measuring the fluorescent signal generated when the encapsulated GFP is released and reacted with the externally added fragment of the split GFP. The system exhibited a limit of detection of 0.17 μg/ml for LLO toxin and 10 CFU/mL for L. monocytogenes with high sensitivity and specificity and no cross-reactivity with other bacteria. The PoreGlow system is practical, rapid, and does not require sample pre-treatment, making it a promising tool for the early detection of L. monocytogenes in food products, which is crucial for preventing outbreaks and protecting public health.

Simulation-guided engineering of split GFPs with efficient β-strand photodissociation

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. To reduce the search space, key states in the dissociation process are modeled by combining classical and enhanced sampling molecular dynamics with QM/MM calculations, enabling the rational design and engineering of split GFPs with up to 20-fold faster photodissociation rates using non-intuitive amino acid changes. This demonstrates the feasibility of modeling complex molecular processes using state-of-the-art computational methods, and the potential of integrating computational methods to increase the success rate in protein engineering projects.

N-linked glycosylation of flavivirus E protein contributes to viral particle formation.

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. The number of secreted subviral particles (SVPs) was significantly reduced in N154A, a glycosylation-null mutant, but increased in D67N, a mutant containing additional glycosylation sites, indicating that the amount of E glycosylation regulates the release of SVPs. SVP secretion was reduced in cells deficient in galactose, sialic acid, and N-acetylglucosamine modifications in the Golgi apparatus; however, these reductions were not significant, suggesting that glycosylation mainly plays a role in pre-Golgi transport. Fluorescent labeling of SVPs using a split green fluorescent protein (GFP) system and time-lapse imaging by retention using selective hooks (RUSH) system revealed that the glycosylation-deficient mutant was arrested before endoplasmic reticulum (ER)- Golgi transport. However, the absence of ERGIC-53 and ERGIC-L, ER-Golgi transport cargo receptors that recognize sugar chains on cargo proteins, does not impair SVP secretion. In contrast, the solubility of the N154A mutant of E or the N15A/T17A mutant of prM in cells was markedly lower than that of the wild type, and proteasome-mediated rapid degradation of these mutants was observed, indicating the significance of glycosylation of both prM and E in proper protein folding and assembly of viral particles in the ER.

A precise and efficient circular RNA synthesis system based on a ribozyme derived from Tetrahymena thermophila.

Classic strategies for circular RNA (circRNA) preparation always introduce large numbers of linear transcripts or extra nucleotides to the circularized product. In this study, we aimed to develop an efficient system for circRNA preparation based on a self-splicing ribozyme derived from an optimized Tetrahymena thermophila group Ⅰ intron. The target RNA sequence was inserted downstream of the ribozyme and a complementary antisense region was added upstream of the ribozyme to assist cyclization. Then, we compared the circularization efficiency of ribozyme or flanking intronic complementary sequence (ICS)-mediated methods through the DNMT1, CDR1as, FOXO3, and HIPK3 genes and found that the efficiency of our system was remarkably higher than that of flanking ICS-mediated method. Consequently, the circularized products mediated by ribozyme are not introduced with additional nucleotides. Meanwhile, the overexpressed circFOXO3 maintained its biological functions in regulating cell proliferation, migration, and apoptosis. Finally, a ribozyme-based circular mRNA expression system was demonstrated with a split green fluorescent protein (GFP) using an optimized Coxsackievirus B3 (CVB3) internal ribosome entry site (IRES) sequence, and this system achieved successful translation of circularized mRNA. Therefore, this novel, convenient, and rapid engineering RNA circularization system can be applied for the functional study and large-scale preparation of circular RNA in the future.

Biosensor-guided rapid screening for improved recombinant protein secretion in Pichia pastoris

Pichia pastoris (Komagataella phaffii) is widely used for industrial production of heterologous proteins due to high secretory capabilities but selection of highly productive engineered strains remains a limiting step. Despite availability of a comprehensive molecular toolbox for construct design and gene integration, there is high clonal variability among transformants due to frequent multi-copy and off-target random integration. Therefore, functional screening of several hundreds of transformant clones is essential to identify the best protein production strains. Screening methods are commonly based on deep-well plate cultures with analysis by immunoblotting or enzyme activity assays of post-induction samples, and each heterologous protein produced may require development of bespoke assays with multiple sample processing steps. In this work, we developed a generic system based on a P. pastoris strain that uses a protein-based biosensor to identify highly productive protein secretion clones from a heterogeneous set of transformants. The biosensor uses a split green fluorescent protein where the large GFP fragment (GFP1-10) is fused to a sequence-specific protease from Tobacco Etch Virus (TEV) and is targeted to the endoplasmic reticulum. Recombinant proteins targeted for secretion are tagged with the small fragment of the split GFP (GFP11). Recombinant protein production can be measured by monitoring GFP fluorescence, which is dependent on interaction between the large and small GFP fragments. The reconstituted GFP is cleaved from the target protein by TEV protease, allowing for secretion of the untagged protein of interest and intracellular retention of the mature GFP. We demonstrate this technology with four recombinant proteins (phytase, laccase, β-casein and β-lactoglobulin) and show that the biosensor directly reports protein production levels that correlate with traditional assays. Our results confirm that the split GFP biosensor can be used for facile, generic, and rapid screening of P. pastoris clones to identify those with the highest production levels.

In Vivo Protein-Protein Binding Competition Assay Based on Split-GFP Reassembly: Proof of Concept.

The split-green fluorescent protein (GFP) reassembly assay is a well-established approach to study protein-protein interactions (PPIs). In this assay, when two interacting proteins X and Y, respectively fused to residues 1-157 and to residues 158-237 of GFP, are co-expressed in E. coli, the two GFP halves are brought to sufficient proximity to reassociate and fold to recreate the functional GFP. At constant protein expression level, the intensity of fluorescence produced by the bacteria is proportional to the binding affinity of X to Y. We hypothesized that adding a third partner (Z) endowed with an affinity for either X or Y would lead to an in vivo competition assay. We report here the different steps of the set-up of this competition assay, and define the experimental conditions required to obtained reliable results. Results show that this competition assay is a potentially interesting tool for screening libraries of binding inhibitors, Z being either a protein or a chemical reagent.

RNA Aptamer-Based Approach to Inhibiting Split-GFP Reconstruction and the Loss of Inhibitory Activity Using Complementary RNA

Abstract Although many studies on protein–protein interactions (PPIs) have been conducted and the importance of PPIs in biological processes has been reported, there is still no versatile research approach that enables us to draw a complete picture of PPIs. One orthodox approach to elucidating the mechanism of each PPI would be to inhibit or enhance the PPI of interest and carefully observe its phenotype. However, since the interaction surfaces of PPIs are generally shallow and wide, it is very difficult to design small molecules that can selectively perturb specific PPIs by interaction with these surfaces. In this report, we adopt reconstruction of split green fluorescence protein (splitGFP) as a model of PPI, and obtained RNA aptamers that bind to one of the components. The reconstitution of splitGFP was inhibited by these aptamers, and this inhibition was cancelled by the addition of their complementary sequences. These processes were monitored by the loss and recovery, respectively, of fluorescence from the reconstructed GFP. The successful development of molecules that reversibly regulate specific PPI is expected to make a significant contribution to life science research.

Rapid prototyping enzyme homologs to improve titer of nicotinamide mononucleotide using a strategy combining cell-free protein synthesis with split GFP.

Engineering biological systems to test new pathway variants containing different enzyme homologs is laborious and time-consuming. To tackle this challenge, a strategy was developed for rapidly prototyping enzyme homologs by combining cell-free protein synthesis (CFPS) with split green fluorescent protein (GFP). This strategy featured two main advantages: (1) dozens of enzyme homologs were parallelly produced by CFPS within hours, and (2) the expression level and activity of each homolog was determined simultaneously by using the split GFP assay. As a model, this strategy was applied to optimize a 3-step pathway for nicotinamide mononucleotide (NMN) synthesis. Ten enzyme homologs from different organisms were selected for each step. Here, the most productive homolog of each step was identified within 24 h rather than weeks or months. Finally, the titer of NMN was increased to 1213 mg/L by improving physiochemical conditions, tuning enzyme ratios and cofactor concentrations, and decreasing the feedback inhibition, which was a more than 12-fold improvement over the initial setup. This strategy would provide a promising way to accelerate design-build-test cycles for metabolic engineering to improve the production of desired products.

Tri-part NanoLuc as a new split technology with potential applications in chemical biology: a mini-review.

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. Inspired by the split green fluorescent protein (GFP) and its β-barrel structure, two split NLuc consisting of peptide fragments have been reported including the binary and ternary NLuc systems. NanoBiT® (large fragment + peptide) has been used extensively. In contrast, tripart split NLuc (large fragment + 2 peptides) has been applied and hardly used, while it has some advantages over NanoBiT in some studies. Nevertheless, tripart NLuc has some drawbacks and challenges to overcome but has several potential characteristics to become a multifunctional and powerful tool. In this review, several aspects of tripart NLuc are studied and a brief comparison with NanoBiT® is given.

Split-GFP Reassembly Assay: Strengths and Caveats from a Multiparametric Analysis.

The split-Green Fluorescent Protein (GFP) reassembly assay is a powerful approach to study protein-protein interactions (PPIs). In this assay, two proteins, respectively, fused to the first seven and the last four β-strands of GFP are co-expressed in E. coli where they can bind to each other, which reconstitutes the full-length GFP. Thus, the fluorescence of the bacteria co-expressing the two fusion proteins accounts for the interaction of the two proteins of interest. The first split-GFP reassembly assay was devised in the early 2000s in Regan's lab. During the last ten years, we have been extensively using this assay to study the interactions of an intrinsically disordered protein (IDP) with two globular partners. Over that period, in addition to accumulating molecular information on the specific interactions under study, we progressively modified the original technique and tested various parameters. In those previous studies, however, we focused on the mechanistic insights provided by the approach, rather than on the method itself. Since methodological aspects deserve attention and the best bipartite reporter to study PPIs involving IDPs remains to be identified, we herein focus on technical aspects. To this end, we first revisit our previous modifications of the original method and then investigate the impact of a panel of additional parameters. The present study unveiled a few critical parameters that deserve consideration to avoid pitfalls and obtain reliable results.

Ebola and Marburg virus VP35 coiled-coil validated as antiviral target by tripartite split-GFP complementation

Ebola virus (EBOV) and Marburg virus (MARV) are highly pathogenic viruses in humans, against which approved antivirals are lacking. During EBOV and MARV infection, coiled-coil mediated oligomerization is essential for the virion protein 35 (VP35) polymerase co-factor function and type I interferon antagonism, making VP35 coiled-coil an elective drug target. We established a tripartite split-green fluorescent protein (GFP) fluorescence complementation (FC) system based on recombinant GFP-tagged EBOV and MARV VP35, which probes VP35 coiled-coil assembly by monitoring fluorescence on E.coli colonies, or invitro in 96/384-multiwell. Oligomerization-defective VP35 mutants showed that correct coiled-coil knobs-into-holes pairing within VP35 oligomer is pre-requisite for GFP tags and GFP detector to reconstitute fluorescing full-length GFP. The method was validated by screening a small compound library, which identified Myricetin and 4,5,6,7-Tetrabromobenzotriazole as inhibitors of EBOV and MARV VP35 oligomerization-dependent FC with low-micromolar IC50 values. These findings substantiate the VP35 coiled-coil value as antiviral target.

CRISPR-Cas9-Mediated Knock-In Approach to Insert the GFP11 Tag into the Genome of a Human Cell Line.

The protocol in this chapter describes a method to label endogenous proteins using a self-complementing split green fluorescent protein (split GFP1-10/11) in a human cell line. By directly delivering Cas9/sgRNA ribonucleoprotein (RNP) complexes through nucleofection, this protocol allows for the efficient integration of GFP11 into a specific genomic locus via CRISPR-Cas9-mediated homology-directed repair (HDR). We use the GFP11 sequence in the form of a single-stranded DNA (ssDNA) as an HDR template. Because the ssDNA with less than 200 nucleotides used here is commercially synthesized, this approach remains cloning-free. The integration of GFP11 is performed in cells stably expressing GFP1-10, thereby inducing fluorescence reconstitution. Subsequently, such a reconstituted signal is analyzed using fluorescence flow cytometry for estimating knock-in efficiencies and enriching the GFP-positive cell population. Finally, the enriched cells can be visualized using fluorescence microscopy.