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Nature Biotechnology Jan 2006Existing variants of green fluorescent protein (GFP) often misfold when expressed as fusions with other proteins. We have generated a robustly folded version of GFP,...
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.
Topics: Bacterial Proteins; Escherichia coli; Green Fluorescent Proteins; Models, Molecular; Molecular Conformation; Protein Binding; Protein Engineering; Protein Folding; Pyrobaculum; Recombinant Fusion Proteins; Spectrometry, Fluorescence
PubMed: 16369541
DOI: 10.1038/nbt1172 -
Annals of Oncology : Official Journal... Nov 2019Due to the efficacy of tropomyosin receptor kinase (TRK) inhibitor therapy and the recent Food and Drug Administration approval of larotrectinib, it is now clinically... (Review)
Review
Due to the efficacy of tropomyosin receptor kinase (TRK) inhibitor therapy and the recent Food and Drug Administration approval of larotrectinib, it is now clinically important to accurately and efficiently identify patients with neurotrophic TRK (NTRK) fusion-driven cancer. These oncogenic fusions occur when the kinase domain of NTRK1, NTRK2 or NTRK3 fuse with any of a number of N-terminal partners. NTRK fusions are characteristic of a few rare types of cancer, such as secretory carcinoma of the breast or salivary gland and infantile fibrosarcoma, but they are also infrequently seen in some common cancers, such as melanoma, glioma and carcinomas of the thyroid, lung and colon. There are multiple methods for identifying NTRK fusions, including pan-TRK immunohistochemistry, fluorescence in situ hybridisation and sequencing methods, and the advantages and drawbacks of each are reviewed here. While testing algorithms will obviously depend on availability of various testing modalities and economic considerations for each individual laboratory, we propose triaging specimens based on histology and other molecular findings to most efficiently identify tumours harbouring these treatable oncogenic fusions.
Topics: Gene Fusion; Humans; Membrane Glycoproteins; Neoplasms; Oncogene Proteins, Fusion; Receptor Protein-Tyrosine Kinases; Receptor, trkA; Receptor, trkB; Receptor, trkC
PubMed: 31738428
DOI: 10.1093/annonc/mdz384 -
The Journal of Biological Chemistry 2021Within the AGC kinase superfamily, gene fusions resulting from chromosomal rearrangements have been most frequently described for protein kinase C (PKC), with gene...
Within the AGC kinase superfamily, gene fusions resulting from chromosomal rearrangements have been most frequently described for protein kinase C (PKC), with gene fragments encoding either the C-terminal catalytic domain or the N-terminal regulatory moiety fused to other genes. Kinase fusions that eliminate regulatory domains are typically gain of function and often oncogenic. However, several quality control pathways prevent accumulation of aberrant PKC, suggesting that PKC fusions may paradoxically be loss of function. To explore this topic, we used biochemical, cellular, and genome editing approaches to investigate the function of fusions that retain the portion of the gene encoding either the catalytic domain or regulatory domain of PKC. Overexpression studies revealed that PKC catalytic domain fusions were constitutively active but vulnerable to degradation. Genome editing of endogenous genes to generate a cancer-associated PKC fusion resulted in cells with detectable levels of fusion transcript but no detectable protein. Hence, PKC catalytic domain fusions are paradoxically loss of function as a result of their instability, preventing appreciable accumulation of protein in cells. Overexpression of a PKC regulatory domain fusion suppressed both basal and agonist-induced endogenous PKC activity, acting in a dominant-negative manner by competing for diacylglycerol. For both catalytic and regulatory domain fusions, the PKC component of the fusion proteins mediated the effects of the full-length fusions on the parameters examined, suggesting that the partner protein is dispensable in these contexts. Taken together, our findings reveal that PKC gene fusions are distinct from oncogenic fusions and present a mechanism by which loss of PKC function occurs in cancer.
Topics: Animals; Binding Sites; COS Cells; Catalytic Domain; Cell Line, Tumor; Chlorocebus aethiops; Diglycerides; Fluorescence Resonance Energy Transfer; Humans; Loss of Function Mutation; Neoplasms; Phosphorylation; Protein Domains; Protein Kinase C; Protein Kinase C-alpha; Recombinant Fusion Proteins
PubMed: 33617877
DOI: 10.1016/j.jbc.2021.100445 -
Protein Expression and Purification Jan 2022Advances in structural biology have been fueled in part by developing techniques for large-scale heterologous expression and purification of proteins. Nevertheless, this... (Comparative Study)
Comparative Study
Advances in structural biology have been fueled in part by developing techniques for large-scale heterologous expression and purification of proteins. Nevertheless, this step is still a bottleneck in biophysical studies of many proteins. Often, fusion proteins are used to increase expression levels, solubility, or both. Here, we compare a recently reported fusion tag, NT*, with Maltose Binding Protein (MBP), a well-known fusion tag and solubility enhancer. NT* shows high expression and solubility when used as an N-terminal fusion partner for several aggregation-prone peptides. Its efficacy in enhancing the solubility of aggregation-prone globular proteins has, however, not been tested. We find here that although the overall expression levels for NT* fusions are much higher than those for the MBP fusion, MBP was far superior for enhancing the solubility of the passenger protein. Nevertheless, the effective yield after purification from the soluble fraction of both MBP-fusion and NT*-fusion was comparable, mainly due to higher expression levels in NT*-fusion and a smaller fraction of the passenger protein net weight being locked in the fusion protein. We conclude that NT* is an excellent fusion tag to improve the overall expression of globular proteins but does not increase the passenger protein's solubility compared to MBP. Proteins that are partially soluble or can be refolded in-vitro will significantly benefit from N-terminal NT* fusions. MBP, however, still remains one of the very few options for an N-terminal fusion if the solubility of the protein after expression is critical for preserving its proper fold or activity.
Topics: Cloning, Molecular; Dual-Specificity Phosphatases; Endopeptidases; Escherichia coli; Gene Expression; Green Fluorescent Proteins; Histidine; Humans; Maltose-Binding Proteins; Mitogen-Activated Protein Kinase Phosphatases; Oligopeptides; Plasmids; Protein Folding; Recombinant Fusion Proteins; Solubility; Tetrahydrofolate Dehydrogenase
PubMed: 34628000
DOI: 10.1016/j.pep.2021.105991 -
Current Pharmaceutical Design 2004A wide range of peptides and polypeptides can be appended to either the N- or C-terminus of G protein-coupled receptors without disrupting substantially ligand binding... (Review)
Review
A wide range of peptides and polypeptides can be appended to either the N- or C-terminus of G protein-coupled receptors without disrupting substantially ligand binding and signal transduction. Following fusion of fluorescent proteins, reporter gene constructs or G protein alpha subunits to the C-terminal tail of a receptor high content and G protein activation assays can be employed to identify agonist ligands. Further modification of the receptor fusions to introduce enhanced levels of constitutive activity and to physically destabilise the protein allows antagonist/inverse agonists screens to be developed in parallel. Equivalent C-terminal addition of pairs of complementary, non-functional, polypeptide fragments allows the application of enzyme complementation techniques. Introduction of N-terminal tags to receptors has also allowed the introduction of novel assay techniques based on a pH-sensitive cyanine dye. These have the capacity to overcome certain limitations of GPCR-fluorescent protein fusions.
Topics: Animals; Drug Design; Green Fluorescent Proteins; Humans; Ligands; Protein Subunits; Receptors, G-Protein-Coupled; Recombinant Fusion Proteins
PubMed: 15279540
DOI: 10.2174/1381612043384295 -
Journal of Virology Jan 2018Fluorescent protein fusions to herpesvirus capsids have proven to be a valuable method to study virus particle transport in living cells. Fluorescent protein fusions to...
Fluorescent protein fusions to herpesvirus capsids have proven to be a valuable method to study virus particle transport in living cells. Fluorescent protein fusions to the amino terminus of small capsid protein VP26 are the most widely used method to visualize pseudorabies virus (PRV) and herpes simplex virus (HSV) particles in living cells. However, these fusion proteins do not incorporate to full occupancy and have modest effects on virus replication and pathogenesis. Recent cryoelectron microscopy studies have revealed that herpesvirus small capsid proteins bind to capsids via their amino terminus, whereas the carboxy terminus is unstructured and therefore may better tolerate fluorescent protein fusions. Here, we describe a new recombinant PRV expressing a carboxy-terminal VP26-mCherry fusion. Compared to previously characterized viruses expressing amino-terminal fusions, this virus expresses more VP26 fusion protein in infected cells and incorporates more VP26 fusion protein into virus particles, and individual virus particles exhibit brighter red fluorescence. We performed single-particle tracking of fluorescent virus particles in primary neurons to measure anterograde and retrograde axonal transport, demonstrating the usefulness of this novel VP26-mCherry fusion for the study of viral intracellular transport. Alphaherpesviruses are among the very few viruses that are adapted to invade the mammalian nervous system. Intracellular transport of virus particles in neurons is important, as this process underlies both mild peripheral nervous system infection and severe spread to the central nervous system. VP26, the small capsid protein of HSV and PRV, was one of the first herpesvirus proteins to be fused to a fluorescent protein. Since then, these capsid-tagged virus mutants have become a powerful tool to visualize and track individual virus particles. Improved capsid tags will facilitate fluorescence microscopy studies of virus particle intracellular transport, as a brighter particle will improve localization accuracy of individual particles and allow for shorter exposure times, reducing phototoxicity and improving the time resolution of particle tracking in live cells.
Topics: Axonal Transport; Capsid Proteins; Cells, Cultured; Cryoelectron Microscopy; Herpesvirus 1, Suid; Luminescent Proteins; Microscopy, Fluorescence; Molecular Structure; Neurons; Recombinant Fusion Proteins; Virus Replication; Red Fluorescent Protein
PubMed: 29046447
DOI: 10.1128/JVI.01193-17 -
Nucleic Acids Research Jun 2020Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recruit heterologous proteins to their sites of action. SSBs perform these...
Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recruit heterologous proteins to their sites of action. SSBs perform these essential functions through a modular structural architecture: the N-terminal domain comprises a DNA binding/tetramerization element whereas the C-terminus forms an intrinsically disordered linker (IDL) capped by a protein-interacting SSB-Ct motif. Here we examine the activities of SSB-IDL fusion proteins in which fluorescent domains are inserted within the IDL of Escherichia coli SSB. The SSB-IDL fusions maintain DNA and protein binding activities in vitro, although cooperative DNA binding is impaired. In contrast, an SSB variant with a fluorescent protein attached directly to the C-terminus that is similar to fusions used in previous studies displayed dysfunctional protein interaction activity. The SSB-IDL fusions are readily visualized in single-molecule DNA replication reactions. Escherichia coli strains in which wildtype SSB is replaced by SSB-IDL fusions are viable and display normal growth rates and fitness. The SSB-IDL fusions form detectible SSB foci in cells with frequencies mirroring previously examined fluorescent DNA replication fusion proteins. Cells expressing SSB-IDL fusions are sensitized to some DNA damaging agents. The results highlight the utility of SSB-IDL fusions for biochemical and cellular studies of genome maintenance reactions.
Topics: DNA Damage; DNA Repair; DNA Replication; DNA, Single-Stranded; DNA-Binding Proteins; Escherichia coli; Fluorescence; Genome, Bacterial; Intrinsically Disordered Proteins; Protein Binding; Recombinant Fusion Proteins; SOS Response, Genetics
PubMed: 32374866
DOI: 10.1093/nar/gkaa320 -
Viruses Nov 2015In the nearly two decades since the popularization of green fluorescent protein (GFP), fluorescent protein-based methodologies have revolutionized molecular and cell... (Review)
Review
In the nearly two decades since the popularization of green fluorescent protein (GFP), fluorescent protein-based methodologies have revolutionized molecular and cell biology, allowing us to literally see biological processes as never before. Naturally, this revolution has extended to virology in general, and to the study of alpha herpesviruses in particular. In this review, we provide a compendium of reported fluorescent protein fusions to herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV) structural proteins, discuss the underappreciated challenges of fluorescent protein-based approaches in the context of a replicating virus, and describe general strategies and best practices for creating new fluorescent fusions. We compare fluorescent protein methods to alternative approaches, and review two instructive examples of the caveats associated with fluorescent protein fusions, including describing several improved fluorescent capsid fusions in PRV. Finally, we present our future perspectives on the types of powerful experiments these tools now offer.
Topics: Alphaherpesvirinae; Biomedical Research; Host-Pathogen Interactions; Luminescent Proteins; Recombinant Fusion Proteins; Staining and Labeling; Virology
PubMed: 26610544
DOI: 10.3390/v7112915 -
Photochemistry and Photobiology Mar 2017Calcium-activated photoproteins, such as aequorin, have been used as luminescent Ca indicators since 1967. After the cloning of aequorin in 1985, microinjection was... (Review)
Review
Calcium-activated photoproteins, such as aequorin, have been used as luminescent Ca indicators since 1967. After the cloning of aequorin in 1985, microinjection was substituted by its heterologous expression, which opened the way for a widespread use. Molecular fusion of green fluorescent protein (GFP) to aequorin recapitulated the nonradiative energy transfer process that occurs in the jellyfish Aequorea victoria, from which these two proteins were obtained, resulting in an increase of light emission and a shift to longer wavelength. The abundance and location of the chimera are seen by fluorescence, whereas its luminescence reports Ca levels. GFP-aequorin is broadly used in an increasing number of studies, from organelles and cells to intact organisms. By fusing other fluorescent proteins to aequorin, the available luminescence color palette has been expanded for multiplexing assays and for in vivo measurements. In this report, we will attempt to review the various photoproteins available, their reported fusions with fluorescent proteins and their biological applications to image Ca dynamics in organelles, cells, tissue explants and in live organisms.
Topics: Aequorin; Calcium; Energy Transfer; Luminescent Proteins; Protein Engineering; Recombinant Fusion Proteins
PubMed: 27925224
DOI: 10.1111/php.12682 -
Protein Expression and Purification Dec 1998For affinity-chromatography-based purification of proteins that are prone to abnormal termination of translation or that may not be modified at their N-termini, affinity...
For affinity-chromatography-based purification of proteins that are prone to abnormal termination of translation or that may not be modified at their N-termini, affinity tags are needed which can be fused to the C-terminus. In this publication we describe that maltose binding protein (MBP) fused to the C-terminus of the plant photoreceptor phytochrome B allows purification of the fusion protein via amylose affinity chromatography. After overexpression in yeast a 125-fold enrichment could be achieved. The spectral properties of phytochrome B were not impaired by the fusion and purification. These results demonstrate that not only the widely used N-terminal fusions of MBP but also C-terminal fusions can be employed for protein purification.
Topics: Affinity Labels; Amylose; Bacterial Proteins; Carrier Proteins; Chromatography, Affinity; Genetic Vectors; Maltose; Maltose-Binding Proteins; Photoreceptor Cells; Phytochrome; Phytochrome B; Plants, Toxic; Protein Binding; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Spectrophotometry; Nicotiana; Transcription Factors
PubMed: 9882570
DOI: 10.1006/prep.1998.0969