ABSTRACT
Autophagy is a critical mechanism deployed by eukaryotic cells in response to stress, including viral infection, to boost the innate antimicrobial responses. However, an increasing number of pathogens hijack the autophagic machinery to facilitate their own replication. Influenza A virus (IAV), responsible for several global pandemics, has an intricate dependence on autophagy for successful replication in mammalian cells. To elucidate key chokepoints in the host stress responses facilitating IAV replication, we constructed a meta-transcriptome of IAV and host gene expression dynamics during early (1-3 hpi), mid (4-6 hpi), and late (8-12 hpi) stages of the viral replication cycle at two multiplicities of infection (MOI): 1 and 5. We supplemented the global transcriptome study with phosphoproteomic analysis of stress-activated protein kinase (SAPK/JNK) signaling in lung carcinoma (predominantly used as an in vitro model of IAV replication) and normal human bronchial epithelial cells. We report significant differences in the activation profiles of autophagy regulating genes upon IAV infection at the two MOI as well as divergent dependence on ULK1 signaling within the normal and cancer cells. Regardless of the cell model, JNK-Thr187 signaling was crucial for the production of infectious viral particles.
Subject(s)
Influenza A virus , Animals , Autophagy/genetics , Epithelial Cells , Humans , Influenza A virus/genetics , Influenza A virus/metabolism , Mammals , Signal Transduction , Virus Replication/geneticsABSTRACT
BACKGROUND: Type I polyketide synthases (PKSs) are multifunctional enzymes responsible for the biosynthesis of a group of diverse natural compounds with biotechnological and pharmaceutical interest called polyketides. The diversity of polyketides is impressive despite the limited set of catalytic domains used by PKSs for biosynthesis, leading to considerable interest in deciphering their structure-function relationships, which is challenging due to high intrinsic flexibility. Among nineteen polyketide synthases encoded by the genome of Mycobacterium tuberculosis, Pks13 is the condensase required for the final condensation step of two long acyl chains in the biosynthetic pathway of mycolic acids, essential components of the cell envelope of Corynebacterineae species. It has been validated as a promising druggable target and knowledge of its structure is essential to speed up drug discovery to fight against tuberculosis. RESULTS: We report here a quasi-atomic model of Pks13 obtained using small-angle X-ray scattering of the entire protein and various molecular subspecies combined with known high-resolution structures of Pks13 domains or structural homologues. As a comparison, the low-resolution structures of two other mycobacterial polyketide synthases, Mas and PpsA from Mycobacterium bovis BCG, are also presented. This study highlights a monomeric and elongated state of the enzyme with the apo- and holo-forms being identical at the resolution probed. Catalytic domains are segregated into two parts, which correspond to the condensation reaction per se and to the release of the product, a pivot for the enzyme flexibility being at the interface. The two acyl carrier protein domains are found at opposite sides of the ketosynthase domain and display distinct characteristics in terms of flexibility. CONCLUSIONS: The Pks13 model reported here provides the first structural information on the molecular mechanism of this complex enzyme and opens up new perspectives to develop inhibitors that target the interactions with its enzymatic partners or between catalytic domains within Pks13 itself.
Subject(s)
Mycobacterium tuberculosis , Polyketides , Bacterial Proteins/metabolism , Mycobacterium tuberculosis/genetics , Mycolic Acids/chemistry , Mycolic Acids/metabolism , Polyketide Synthases/chemistry , Polyketide Synthases/genetics , Polyketide Synthases/metabolism , Polyketides/metabolismABSTRACT
Fluorescent proteins (FPs) are versatile biomarkers that facilitate effective detection and tracking of macromolecules of interest in real time. Engineered FPs 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 FP from a species of strawberry anemone, Corynactis californica, delivered pairs of fragments with up to threefold faster complementation than split GFP. We present the biophysical, biochemical, and structural characteristics of five full-length variants derived from these split C. californica GFP (ccGFP). These ccGFP variants are more tolerant under chemical denaturation with up to 8 kcal/mol lower unfolding free energy than that of the sfGFP. It is likely that some of these ccGFP variants could be suitable as biomarkers under more adverse environments where sfGFP fails to survive. A structural analysis suggests explanations of the variations in stabilities among the ccGFP variants.
Subject(s)
Green Fluorescent Proteins , Green Fluorescent Proteins/chemistry , BiomarkersABSTRACT
A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP ß-strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was tested by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10-11) hairpin in complex with GFP(1-9) was determined at a resolution of 2.6â Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10-11) hairpin with a variety of GFP(1-9) mutants engineered for favorable crystallization.
Subject(s)
Crystallization/methods , Green Fluorescent Proteins/chemistry , Luminescent Proteins/chemistry , Recombinant Fusion Proteins/chemistry , Amino Acid Sequence , Crystallography, X-Ray/methods , Gene Expression , Green Fluorescent Proteins/metabolism , Luminescent Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Protein Folding , Recombinant Fusion Proteins/metabolism , Red Fluorescent ProteinABSTRACT
Exploring the function and 3D space of large multidomain protein targets often requires sophisticated experimentation to obtain the targets in a form suitable for structure determination. Screening methods capable of selecting well-expressed, soluble fragments from DNA libraries exist, but require the use of automation to maximize chances of picking a few good candidates. Here, we describe the use of an insertion dihydrofolate reductase (DHFR) vector to select in-frame fragments and a split-GFP assay technology to filter-out constructs that express insoluble protein fragments. With the incorporation of an IPCR step to create high density, focused sublibraries of fragments, this cost-effective method can be performed manually with no a priori knowledge of domain boundaries while permitting single amino acid resolution boundary mapping. We used it on the well-characterized p85α subunit of the phosphoinositide-3-kinase to demonstrate the robustness and efficiency of our methodology. We then successfully tested it onto the polyketide synthase PpsC from Mycobacterium tuberculosis, a potential drug target involved in the biosynthesis of complex lipids in the cell envelope. X-ray quality crystals from the acyl-transferase (AT), dehydratase (DH) and enoyl-reductase (ER) domains have been obtained.
Subject(s)
Gene Library , Protein Structure, Tertiary , Class Ia Phosphatidylinositol 3-Kinase/chemistry , Class Ia Phosphatidylinositol 3-Kinase/genetics , Crystallography, X-Ray , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Mycobacterium tuberculosis/enzymology , Polyketide Synthases/chemistry , Polyketide Synthases/genetics , Polymerase Chain Reaction , Solubility , Tetrahydrofolate Dehydrogenase/geneticsABSTRACT
The pathogen Brucella melitensis secretes a Toll/interleukin-1 receptor (TIR) domain containing protein that abrogates host innate immune responses. In this study, we have characterized the biochemical interactions of Brucella TIR-like protein TcpB with host innate immune adaptor proteins. Using protein-fragment complementation assays based on Gaussia luciferase and green fluorescent protein, we find that TcpB interacts directly with MyD88 and that this interaction is significantly stronger than the interaction of TcpB with TIRAP, the only other adaptor protein that detectably interacts with TcpB. Surprisingly, the TcpB-MyD88 interaction depends on the death domain (DD) of MyD88, and TcpB does not interact with the isolated TIR domain of MyD88. TcpB disrupts MyD88(DD)-MyD88(DD), MyD88(DD)-MyD88(TIR) and MyD88(DD)-MyD88 interactions but not MyD88-MyD88 or MyD88(TIR)-MyD88(TIR) interactions. Structural models consistent with these results suggest how TcpB might inhibit TLR signaling by targeting MyD88 via a DD-TIR domain interface.
Subject(s)
Brucella melitensis/metabolism , Myeloid Differentiation Factor 88/metabolism , Receptors, Interleukin-1/metabolism , Animals , CHO Cells , Cricetinae , Green Fluorescent Proteins/chemistry , HEK293 Cells , Humans , Luciferases/chemistry , Myeloid Differentiation Factor 88/chemistry , Protein Structure, Tertiary , Receptors, Interleukin-1/chemistryABSTRACT
Here, we describe the isolation of 18 unique anti SARS-CoV-2 human single-chain antibodies from an antibody library derived from healthy donors. The selection used a combination of phage and yeast display technologies and included counter-selection strategies meant to direct the selection of the receptor-binding motif (RBM) of SARS-CoV-2 spike protein's receptor binding domain (RBD2). Selected antibodies were characterized in various formats including IgG, using flow cytometry, ELISA, high throughput SPR, and fluorescence microscopy. We report antibodies' RBD2 recognition specificity, binding affinity, and epitope diversity, as well as ability to block RBD2 binding to the human receptor angiotensin-converting enzyme 2 (ACE2) and to neutralize authentic SARS-CoV-2 virus infection in vitro. We present evidence supporting that: 1) most of our antibodies (16 out of 18) selectively recognize RBD2; 2) the best performing 8 antibodies target eight different epitopes of RBD2; 3) one of the pairs tested in sandwich assays detects RBD2 with sub-picomolar sensitivity; and 4) two antibody pairs inhibit SARS-CoV-2 infection at low nanomolar half neutralization titers. Based on these results, we conclude that our antibodies have high potential for therapeutic and diagnostic applications. Importantly, our results indicate that readily available non immune (naïve) antibody libraries obtained from healthy donors can be used to select high-quality monoclonal antibodies, bypassing the need for blood of infected patients, and offering a widely accessible and low-cost alternative to more sophisticated and expensive antibody selection approaches (e.g. single B cell analysis and natural evolution in humanized mice).
Subject(s)
Antibodies, Viral , COVID-19 , Single-Chain Antibodies , Antibodies, Neutralizing , COVID-19/immunology , Epitopes , Humans , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolismABSTRACT
In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.
Subject(s)
Chemical Fractionation/methods , Chemistry, Physical/methods , Protein Engineering/methods , Proteomics/methods , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
Split green fluorescent protein (GFP) has been used in a panoply of cellular biology applications to study protein translocation, monitor protein solubility and aggregation, detect protein-protein interactions, enhance protein crystallization, and even map neuron contacts. Recent work shows the utility of split fluorescent proteins for large scale labeling of proteins in cells using CRISPR, but sets of efficient split fluorescent proteins that do not cross-react are needed for multiplexing experiments. We present a new monomeric split green fluorescent protein (ccGFP) engineered from a tetrameric GFP found in Corynactis californica, a bright red colonial anthozoan similar to sea anemones and scleractinian stony corals. Split ccGFP from C. californica complements up to threefold faster compared to the original Aequorea victoria split GFP and enable multiplexed labeling with existing A. victoria split YFP and CFP.
Subject(s)
Anthozoa/metabolism , Green Fluorescent Proteins/metabolism , Protein Engineering/methods , Animals , Anthozoa/genetics , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/standards , Protein DomainsABSTRACT
In vitro display technologies based on phage and yeast have a successful history of selecting single-chain variable fragment (scFv) antibodies against various targets. However, single-chain antibodies are often unstable and poorly expressed in Escherichia coli. Here, we explore the feasibility of converting scFv antibodies to an intrinsically fluorescent format by inserting the monomeric, stable fluorescent protein named thermal green, between the light- and heavy-chain variable regions. Our results show that the scTGP format maintains the affinity and specificity of the antibodies, improves expression levels, allows one-step fluorescent assay for detection of binding and is a suitable reagent for epitope binning. We also report the crystal structure of an scTGP construct that recognizes phosphorylated tyrosine on FcεR1 receptor of the allergy pathway.
Subject(s)
Single-Chain Antibodies , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/genetics , Fluorescent DyesABSTRACT
Protein production in Escherichia coli involves high-level expression in a culture, followed by harvesting of the cells and finally their disruption, or lysis, to release the expressed proteins. We compare three high-throughput chemical lysis methods to sonication, using a robotic platform and methodologies developed in our laboratory [1]. Under the same expression conditions, all lysis methods varied in the degree of released soluble proteins. With a set of 96 test proteins, we used our split GFP to quantify the soluble and insoluble protein fractions after lysis. Both the amount of soluble protein and the percentage recovered in the soluble fraction using SoluLyse were well correlated with sonication. Two other methods, Bugbuster and lysozyme, did not correlate well with sonication. Considering the effects of lysis methods on protein solubility is especially important when accurate protein solubility measurements are needed, for example, when testing adjuvants, growth media, temperature, or when establishing the effects of truncation or sequence variation on protein stability.
Subject(s)
Escherichia coli/genetics , Escherichia coli/metabolism , Green Fluorescent Proteins/chemistry , Proteins/chemistry , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Polyketide Synthases/genetics , Polyketide Synthases/metabolism , Proteins/genetics , Proteins/metabolism , Solubility , Temperature , Transferases (Other Substituted Phosphate Groups)ABSTRACT
Interactions between the cytoplasmic domains of viral transmembrane proteins and host machinery often determine the outcome of viral infection. The M2 protein of influenza A has been identified as a key player in autophagy-mediated viral replication. Here, we describe the engineering and validation of an antibody specific for the cytoplasmic domain of the M2 protein. Through phage and yeast display selection techniques, we obtained an antibody that recognizes: 1) the M2 cytoplasmic domain purified from bacterial inclusion bodies and refolded, 2) full-length M2 recombinant protein expressed in mammalian cells, and 3) native M2 protein in influenza A infected cells. This antibody can serve as a molecular tool to enhance our knowledge of protein-protein interactions between influenza A virus and the host cell machinery. We anticipate the methods described herein will further the development of antibodies specific to the cytoplasmic domains of transmembrane proteins.
Subject(s)
Antibodies, Viral/immunology , Antibodies/immunology , Influenza A virus/immunology , Influenza, Human , Viral Matrix Proteins/immunology , HEK293 Cells , HumansABSTRACT
Overproduction of soluble and stable proteins for functional and structural studies is a major bottleneck for structural genomics programs and traditional biochemistry laboratories. Many high-payoff proteins that are important in various biological processes are "difficult to handle" as protein reagents in their native form. We have recently made several advances in enabling biochemical technologies for improving protein stability (http://www.lanl.gov/projects/gfp/), allowing stratagems for efficient protein domain trapping, solubility-improving mutations, and finding protein folding partners. In particular split-GFP protein tags are a very powerful tool for detection of stable protein domains. Soluble, stable proteins tagged with the 15 amino acid GFP fragment (amino acids 216-228) can be detected in vivo and in vitro using the engineered GFP 1-10 "detector" fragment (amino acids 1-215). If the small tag is accessible, the detector fragment spontaneously binds resulting in fluorescence. Here, we describe our current and on-going efforts to move this process from the bench (manual sample manipulation) to an automated, high-throughput, liquid-handling platform. We discuss optimization and validation of bacterial culture growth, lysis protocols, protein extraction, and assays of soluble and insoluble protein in multiple 96 well plate format. The optimized liquid-handling protocol can be used for rapid determination of the optimal, compact domains from single ORFS, collections of ORFS, or cDNA libraries.
Subject(s)
Proteins/chemistry , Proteomics/methods , Robotics/methods , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Green Fluorescent Proteins/analysis , Protein Engineering/methods , Protein Folding , Proteins/genetics , Proteins/metabolism , SolubilityABSTRACT
Existing variants of green fluorescent protein (GFP) often misfold when expressed as fusions with other proteins. We have generated a robustly folded version of GFP, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides. Compared to 'folding reporter' GFP, a folding-enhanced GFP containing the 'cycle-3' mutations and the 'enhanced GFP' mutations F64L and S65T, superfolder GFP shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics. The fluorescence of Escherichia coli cells expressing each of eighteen proteins from Pyrobaculum aerophilum as fusions with superfolder GFP was proportional to total protein expression. In contrast, fluorescence of folding reporter GFP fusion proteins was strongly correlated with the productive folding yield of the passenger protein. X-ray crystallographic structural analyses helped explain the enhanced folding of superfolder GFP relative to folding reporter GFP.
Subject(s)
Escherichia coli/genetics , Escherichia coli/metabolism , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/metabolism , Protein Engineering/methods , Pyrobaculum/metabolism , Bacterial Proteins/analysis , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Models, Molecular , Molecular Conformation , Protein Binding , Protein Folding , Pyrobaculum/genetics , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Spectrometry, Fluorescence/methodsABSTRACT
The identification of soluble, folded domains of proteins is a recurring task in modern molecular biology. We detail a protocol for identifying compact soluble protein domains using a self-assembling two-part split-GFP comprised of a detector fragment (GFP ß-strands 1 through 10, or GFP1-10) and a tagging fragment (GFP ß-strand 11, or GFP11). The assay is performed in E. coli cells and in cell extracts. A selection step insures the protein fragments are in frame and contain no stop codons, while an inverse PCR is used to enrich protein fragment libraries containing a specific target sequence.
Subject(s)
Codon, Terminator/genetics , Green Fluorescent Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Green Fluorescent Proteins/genetics , Protein Binding , Protein FoldingABSTRACT
Most cellular processes are driven by complex protein-protein interaction networks. Identifying key players and characterizing their interactions at the cellular and molecular level is of key importance to understand biochemical mechanisms that control cellular responses. Here, we detail a protocol for monitoring protein-protein interactions in E. coli cells or in cell extracts using a tripartite split-GFP system comprised of a protein interaction detector fragment (GFP ß-strands 1 through 9 or GFP1-9) and small tagging fragments of GFP ß-strands 10 (GFP10) and 11 (GFP11). Interaction of bait and prey proteins fused to GFP10 and GFP11 tether the small GFP fragments, allowing self-association with GFP1-9. In this scenario, fluorescence intensity of the reconstituted GFP is correlated with the strength of interaction.
Subject(s)
Green Fluorescent Proteins/metabolism , Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Protein Binding , Proteins/chemistry , Proteins/geneticsABSTRACT
Superfolder GFP (sGFP) is a variant of the Green Fluorescent Protein that folds efficiently when fused to poorly folded proteins. In this study, we show that sGFP, but not enhanced GFP, is functional in vivo at 70 degrees C in the extreme thermophile Thermus thermophilus (Tth); thus, permitting the use of sGFP as a localization tag in vivo. We created a suite of plasmids that allow the expression of carboxy-terminal sGFP fusion proteins in both Escherichia coli and Tth. In order to demonstrate the facility of sGFP as an in vivo localization tag in Tth, we tagged GroES (the small subunit of the bacterial GroES/GroEL chaperone), NarC (a membrane component of the nitrate respiration apparatus) and PhoA (a TAT-secreted periplasmic protein), and visualized the distribution of the sGFP fusion proteins using confocal microscopy. Fusions to NarC and PhoA produced enzymatically active proteins that complemented both the narC and the phoA strains respectively. Observation of the distribution of the GroES-sGFP protein by confocal microscopy revealed a homogeneous fluorescence in the cells, which is in full agreement with the cytoplasmic nature of GroES, whereas the NarC-sGFP protein was localized to the membrane. Finally, a combination of confocal microscopy and biochemistry revealed that PhoA is localized in the periplasm. We suggest that sGFP will be broadly applicable in characterizing various extreme thermophile systems.
Subject(s)
Green Fluorescent Proteins/metabolism , Hot Temperature , Microbiological Techniques/methods , Thermus thermophilus/metabolism , Bacterial Proteins/analysis , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Luminescent Proteins/metabolism , Microscopy, Confocal , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Thermus thermophilus/geneticsABSTRACT
Many green fluorescent protein (GFP) variants have been developed for use as fluorescent tags, and recently a superfolder GFP (sfGFP) has been developed as a robust folding reporter. This new variant shows increased stability and improved folding kinetics, as well as 100% recovery of native protein after denaturation. Here, we characterize sfGFP, and find that this variant exhibits hysteresis as unfolding and refolding equilibrium titration curves are non-coincident even after equilibration for more than eight half-lives as estimated from kinetic unfolding and refolding studies. This hysteresis is attributed to trapping in a native-like intermediate state. Mutational studies directed towards inhibiting chromophore formation indicate that the novel backbone cyclization is responsible for the hysteresis observed in equilibrium titrations of sfGFP. Slow equilibration and the presence of intermediates imply a rough landscape. However, de novo folding in the absence of the chromophore is dominated by a smoother energy landscape than that sampled during unfolding and refolding of the post-translationally modified polypeptide.
Subject(s)
Fluorescent Dyes/chemistry , Green Fluorescent Proteins/chemistry , Thermodynamics , Circular Dichroism , Cyclophilin A/chemistry , Cyclophilin A/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Guanidine/chemistry , Kinetics , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Protein Denaturation , Protein Folding , Spectrometry, FluorescenceABSTRACT
For structural studies by x-ray crystallography and nuclear magnetic resonance it is important for the target protein to be available in large quantity and high purity. Escherichia coli expression systems remain the most versatile and convenient means to produce a large quantity of recombinant proteins. Unfortunately, some proteins fail to be expressed in E. coli or are expressed in an insoluble form. To overcome the difficulty of no expression or expression at a very low level, a simple and efficient approach of screening a library of variants of a target protein with randomized N-termini was devised. In this method, a few N-terminal residues are randomized by designing a mixture of oligonucleotides for the forward PCR primer and we fuse the library in front of green fluorescent protein, which serves as a reporter for the target protein expression level and folding yield. In favorable cases this approach can result in high-level soluble expression of recombinant proteins in E. coli. This chapter describes the results of a test of this approach with a bacterial protein (the HI0952 gene product) that is not well expressed in E. coli.
Subject(s)
Bacterial Proteins/metabolism , Escherichia coli/metabolism , Gene Expression , Gene Library , Recombinant Fusion Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Cloning, Molecular , Escherichia coli/genetics , Green Fluorescent Proteins/genetics , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , SolubilityABSTRACT
Existing protein tagging and detection methods are powerful but have drawbacks. Split protein tags can perturb protein solubility or may not work in living cells. Green fluorescent protein (GFP) fusions can misfold or exhibit altered processing. Fluorogenic biarsenical FLaSH or ReASH substrates overcome many of these limitations but require a polycysteine tag motif, a reducing environment and cell transfection or permeabilization. An ideal protein tag would be genetically encoded, would work both in vivo and in vitro, would provide a sensitive analytical signal and would not require external chemical reagents or substrates. One way to accomplish this might be with a split GFP, but the GFP fragments reported thus far are large and fold poorly, require chemical ligation or fused interacting partners to force their association, or require coexpression or co-refolding to produce detectable folded and fluorescent GFP. We have engineered soluble, self-associating fragments of GFP that can be used to tag and detect either soluble or insoluble proteins in living cells or cell lysates. The split GFP system is simple and does not change fusion protein solubility.