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PubMed1999

An integral membrane green fluorescent protein marker, Us9-GFP, is quantitatively retained in cells during propidium iodide-based cell cycle analysis by flow cytometry.

Previously, we described GFP-spectrin, a membrane-localized derivative of the green fluorescent protein that can be employed as a marker during the simultaneous identification of transfected cells and cell cycle analysis by flow cytometry (Kalejta et al., Cytometry 29: 286-291, 1997). A membrane-anchored GFP fusion protein is necessary because the ethanol permeabilization step required to achieve efficient propidium iodide staining allows cytoplasmic GFP to leach out of the cell. However, viable cells expressing GFP-spectrin are not as bright as cells expressing cytoplasmic GFP and their fluorescence intensity is further diminished after ethanol treatment. Here, we demonstrate that the fluorescence intensity of cells expressing an integral membrane GFP fusion protein (Us9-GFP) is similar to that of cells expressing cytoplasmic GFP and is quantitatively maintained in cells after ethanol treatment. By allowing an accurate assessment of the expression level of GFP, Us9-GFP allows a more precise analysis of the effects of a cotransfected plasmid on the cell cycle and thus represents an improvement upon the original membrane-associated GFP fusion proteins employed in this assay.

Cell Cycle↗

PubMed1998

Expression of a chromosomally integrated, single-copy GFP gene in Candida albicans, and its use as a reporter of gene regulation.

Genetically engineered versions of the GFP gene, which encodes the green fluorescent protein of Aequorea victoria, were placed under the control of the constitutively active Candida albicans ACT1 promoter and integrated in single copy into the genome of this pathogenic yeast. Integrative transformants in which one of the two ACT1 alleles had been replaced by a GFP gene exhibited a homogeneous, constitutive fluorescent phenotype. Cells expressing GFP with the wild-type chromophore exhibited very weak fluorescence compared to those GFP proteins with the S65T or S65A, V68L, S72A (GFPmut2) chromophore mutations. Substitution of the CTG codon, which specifies serine instead of leucine in C. albicans, by TTG was absolutely necessary for GFP expression. Although GFP mRNA levels in cells containing a GFP gene with the CTG codon were comparable to those of transformants containing GFP with the TTG substitution, only the latter produced GFP protein, as detected by Western blotting, suggesting that the frequent failure to express heterologous genes in C. albicans is principally due to the noncanonical codon usage. Transformants expressing the modified GFP gene from the promoter of the SAP2 gene, which encodes one of the secreted acid proteinases of C. albicans, showed fluorescence only under conditions which promote proteinase expression, thereby demonstrating the utility of stable, chromosomally integrated GFP reporter genes for the study of gene activation in C. albicans.

Actins↗

PubMed2000

GFP as a Genetic Marker Scorable Throughout the Life Cycle of Transgenic Zebra Fish.

A fish expression vector, FRM, was constructed by fusing the carp beta-actin promoter and first intron to the ocean pout antifreeze protein terminator and putative boundary element. Mutant forms of the green fluorescent protein (GFP) were engineered into this vector, and the resultant series of vectors, FRMwg, FRM3wg (green GFP), and FRM2bl (blue GFP), were used to make transgenic zebra fish. After microinjection of either supercoiled or linearized DNA into one-celled eggs, GFP-expressing cells could be monitored by fluorescence microscopy commencing with the midblastula transition and continuing through embryogenesis. From adult fish, which retained scorable GFP either as patches or as a uniform fluorescence, 11 green and 1 blue GFP-expressing lines of zebra fish have been established. Expression of GFP was nearly ubiquitous and similar among all of these lines. Embryonic expression could be scored at 15 to 30 hours postfertilization and was seen throughout the embryo with the exceptions of the yolk, red blood cells, and in some lines, portions of the head. Adult expression was in a majority of tissues (e.g., muscle, brain, intestine, and heart, but not red blood cells). The notable difference between lines was that fluorescent eggs were scorable in seven of the lines. Adult homozygotes from a different subset of eight lines could be selected by the relative intensity of the GFP marking when compared with that in sibling heterozygotes. All 12 lines contain apparent single locus, multicopy, tandem integrations (1.5-100 copies per cell) of the transgenic DNA. The fish expression vector FRM could be used to drive nearly ubiquitous and strong expression of gene products other than GFP. The GFP expression vectors, FRMwg, FRM2wg, FRM3wg, and FRM2bl, may be useful for optimization of transgenesis and as a comarker. GFP-expressing zebra fish lines could facilitate experimental analysis of chimerism and in vivo gene targeting.

Journal Article↗

PubMed2005

In vivo expression of GFP transgene delivered via a replicating feline leukemia virus.

We previously described replication-competent feline leukemia virus (FeLV) vectors with high-level and stable expression of a green fluorescent protein (GFP) or a suicide transgene in cell cultures in vitro. Considering that FeLV might potentially be used to deliver therapeutic genes in vivo, we first evaluated the expression of the GFP gene introduced in cats by the FeLV, Rickard subgroup A (FRA) construct. Eight newborn kittens were either inoculated with pFRA-GFP plasmid DNA intradermally, or challenged intraperitoneally with FRA-GFP-infected feline fibroblasts. During a 12-week observation period, five cats were shown to be progressively viremic. Quantitative PCR and RT-PCR analyses of plasma and tissue samples from these cats showed that GFP was retained in FeLV DNA or RNA to a variable degree, ranging from 0.002 to 27.890%. Tissue DNA samples were analyzed by PCR for the status of GFP and the env-transgene complex. While the proviruses carrying the GFP transgene were shown to be minor species, all tissues, however, retained the full-length GFP transgene. Despite the occurrence of predominant species with various deletions in the viral genome, approximately 1-3% of the total cell population was GFP-positive in the lymphoid tissues as visualized by laser confocal microscopy. Co-localization of immunofluorescent cells indicated that CD3-positive T cells, dendritic cells and macrophages were the major targets for GFP expression. These findings on the detectable in vivo expression of GFP for as long as a period of 3 months could be viewed positively for contemplating a therapeutic strategy for control of FeLV infection in the cats.

Animals↗

PubMed2006

The use of GFP to localize Rho GTPases in living cells.

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has revolutionized the study of protein localization and dynamics. GFP fusions permit analysis of proteins in living cells and offer distinct advantages over conventional immunofluorescence. Among these are lower background, higher resolution, robust dual color colocalization, and avoidance of fixation artifacts. In the case of Ras and Rho family proteins, GFP fusions have allowed breakthroughs in the understanding of how CAAX proteins are targeted to specific cell membranes and how signaling at different membranes can result in different cellular responses. GFP-tagged Rho proteins have also been informative in analyzing the interactions with the cytosolic chaperone, RhoGDI. The major disadvantages of studying GFP fusion proteins is that they are generally overexpressed relative to endogenous proteins, and the GFP tag can, in principle, affect protein function. Fortunately, in the case of Ras and Rho family proteins, a GFP tag at the N terminus seems to have little effect on protein targeting and function. Nevertheless, it is prudent to confirm GFP fusion protein data with the study of the endogenous protein. This chapter describes the tagging of Rho proteins with GFP and the analysis of GFP-Rho protein localization by epifluorescence and confocal microscopy. It further describes methods of analyzing endogenous Rho proteins as confirmation of data acquired using GFP-Rho fusion proteins. These techniques will be useful for anyone studying Rho protein function and are widely applicable to many cell types and signal transduction systems.

Amino Acid Sequence↗

PubMed2000

Expression and intracellular localization of leptin receptor long isoform-GFP chimera.

The leptin receptor (OBR) and its ligand leptin (OB) are key players in the regulation of body weight. The OBR is a member of the class I cytokine receptor family and is alternatively spliced into at least six different isoforms. The multiple forms are identical in their extracellular and transmembrane regions but differ in lengths. The two predominant isoforms include a long form (OBR(l)) with an intracellular domain of 303 amino acids and a shorter form (OBR(s)) with an intracellular domain of 34 amino acids. We have constructed a recombinant OBR(l) chimera with the green fluorescent protein (GFP) by fusing GFP to the C-terminus of the OBR(l). The OBR(l)-GFP chimera was transiently transfected and expressed in SHSY5Y and HEK293 cells. In a STAT-Luciferase assay we show that the GFP moiety in this chimera did not affect the signalling capacity of OBR(l)-GFP. In both SHSY5Y and HEK293 cells transfected with OBR(l)-GFP, a predominant intracellular green OBR(l)-GFP fluorescence was detected in vesicles also positive for internalized fluorophore conjugated leptin. We also found that treatment with the lysosomotropic reagent monensin did not relocalize OBR(l)-GFP together with the human transferrin receptor in recycling endosomes, indicating OBR(l)-GFP not to participate in this pathway. In biotinylation-streptavidin pulse chase experiments, using antibodies raised against GFP and OBR, we observed that the rate of early appearance of OBR(s) at the cell surface, upon leptin stimulation, was faster than that found for OBR(l)-GFP. Taken together, our results provide novel data concerning the intracellular trafficking of the two different isoforms of the leptin receptor.

Biotinylation↗

PubMed2003

Development and application of unstable GFP variants to kinetic studies of mycobacterial gene expression.

Unstable variants of green fluorescent protein (GFP) tagged with C-terminal extensions, which are targets for a tail specific protease, have been described in Escherichia coli and Pseudomonas putida [Appl. Envir. Microbiol. 64 (1998) 2240]. We investigated whether similar modifications to flow cytometer optimised GFP (GFPmut2) could be used to generate unstable variants of GFP for gene expression studies in mycobacteria. We constructed GFP variants in a mycobacterial shuttle vector under the control of the regulatory region of the inducible Mycobacterium smegmatis acetamidase gene. GFP expression was induced by the addition of acetamide and the stability of the GFP variants in M. smegmatis, following the removal of the inducer to switch off their expression, was determined using spectrofluorometry and flow cytometry. We demonstrate that, compared to the GFPmut2 (half-lives>7 days), the modified GFP variants exhibit much lower half-lives (between 70 and 165 min) in M. smegmatis. To investigate their utility in the measurement of mycobacterial gene expression, we cloned the promoter region of a putative amino acid efflux pump gene, lysE (Rv1986), from Mycobacterium tuberculosis together with the divergently transcribed, putative lysR-type regulator gene (Rv1985c) upstream of one of the unstable GFP variants. We found that the expression kinetics of the lysRE-gfp fusion were identical throughout the M. smegmatis growth curve to those measured using a conventional lysRE-xylE reporter fusion, peaking upon entry into stationary phase. In addition, it was established that the tagged GFP variants were also unstable in Mycobacterium bovis BCG. Thus, we have demonstrated that unstable GFP variants are suitable reporter genes for monitoring transient gene expression in fast- and slow-growing mycobacteria.

Acetamides↗

PubMed2001

Visualizing differences in ligand-induced beta-arrestin-GFP interactions and trafficking between three recently characterized G protein-coupled receptors.

beta-Arrestin 1-GFP or beta-arrestin 2-GFP were coexpressed transiently with G protein-coupled receptor kinase 2 within cells stably expressing the orexin-1, apelin or melanin-concentrating hormone (MCH), receptors. In response to agonist ligands both the orexin-1 and apelin receptors were able to rapidly translocate both beta-arrestin 1-GFP and beta-arrestin 2-GFP from cytoplasm to the plasma membrane. For the MCH receptor this was only observed for beta-arrestin 2-GFP. beta-Arrestin 1-GFP translocated by the apelin receptor remained at the plasma membrane during prolonged exposure to ligand even though the receptor became internalized. By contrast, for the orexin-1 receptor, internalization of beta-arrestin 1-GFP within punctate vesicles could be observed for over 60 min in the continued presence of agonist. Co-internalization of the orexin-1 receptor was observed by monitoring the binding and trafficking of TAMRA-(5- and 6-carboxytetramethylrhodamine) labelled orexin-A. Subsequent addition of an orexin-1 receptor antagonist resulted in cessation of incorporation of beta-arrestin 1-GFP into vesicles at the plasma membrane and a gradual clearance of beta-arrestin 1-GFP from intracellular vesicles. For the melanin-concentrating hormone receptor the bulk of translocated beta-arrestin 2-GFP was maintained at concentrated foci close to, or at, the plasma membrane. These results demonstrate very distinct features of beta-arrestin-GFP interactions and trafficking for three G protein-coupled receptors for which the natural ligands have only recently been identified and which were thus previously considered as orphan receptors.

Animals↗

PubMed2000

A new approach to gene mutation analysis using "GFP-Display".

The unique behavior of green fluorescent protein (GFP) on SDS-PAGE was applied to the detection of a single amino acid substitution in GFP-tagged polypeptides. This simple detection method using SDS/urea gels was designated GFP-display. The N-terminal 18 or 37 amino acids of K-Ras was used as a model GFP-tagged polypeptide. K-ras exon 1 was fused to a gfp cDNA at each end and expressed in Escherichia coli. Amino acid number 12 of K-Ras (wild type; Gly) was changed to Ser, Arg, Cys, Asp, Ala, or Val, and the mobility shift of the greenish fluorescent bands in the SDS/urea gel was analyzed. These mutants were easily detected by GFP-display; however, detection depended strongly on the urea concentration and electrophoresis temperature. Subsequently, GFP-display was applied to the 36 amino acids encoding human p53 exon 7. Amino acid number 248 (wild type; Arg) was changed to Gly, Trp, Gln, Pro, or Leu, and similar mobility shifts were observed. GFP-display could be coupled with an in vitro translation system. Fluorescent active GFP and GFP-Ras fusion proteins were synthesized within a few hours. GFP-display shows potential as a modern approach to gene mutation analysis at the protein level, and is a useful method for protein engineering studies.

DNA Mutational Analysis↗

PubMed1999

Comparison of fixation protocols for adherent cultured cells applied to a GFP fusion protein of the epidermal growth factor receptor.

BACKGROUND: The analysis of the subcellular distribution of proteins is essential for the understanding of processes such as signal transduction. In most cases, the parallel analysis of multiple components requires fixation and immunofluorescence labeling. Therefore, one has to ascertain that the fixation procedure preserves the in vivo protein distribution. Fusion proteins with the green fluorescent protein (GFP) are ideal tools for this purpose. However, one must consider specific aspects of the fluorophore formation or degradation, i.e. reactions that may interfere with the detection of GFP fusion proteins. METHODS: Fusion proteins of the epidermal growth factor receptor (EGFR) with GFP as well as free, soluble GFP stably or transiently expressed in adherent cultured cells served as test cases for comparing the distribution in vivo with that after fixation by conventional epifluorescence and laser scanning microscopy. Indirect immunofluorescence was employed to compare the distributions of the GFP signal and of the GFP polypeptide in the fusion protein. RESULTS: Paraformaldehyde (PFA) fixation with subsequent mounting in the antifading agent Mowiol, but not in Tris- or HEPES buffered saline, led to a partial redistribution of the EGFR from the plasma membrane to the perinuclear region. The redistribution was confirmed with the GFP and EGFR immunofluorescence. The in vivo distribution in Mowiol mounted cells was preserved if cells were treated with a combined PFA/methanol fixation procedure, which also retained the fluorescence of soluble GFP. The anti-GFP antiserum was negative for the N-terminal fusion protein. CONCLUSIONS: The combined PFA/methanol protocol is universally applicable for the fixation of transmembrane and soluble cytoplasmic proteins and preserves the fluorescence of GFP.

Animals↗

PubMed2005

GFP as a tool for the analysis of proteins in the flagellar basal apparatus of Chlamydomonas.

Green fluorescent protein (GFP) was used to analyse three proteins in the flagellar basal apparatus of C. reinhardtii: (1) Striated fiber assemblin (SFA), the major component of the striated microtubule-associated fibers; (2) Centrin, present in the nucleus basal body connectors (NBBCs) and the distal connecting fiber (dCF) between the two basal bodies; and (3) DIP13, the Chlamydomonas homologue of human autoantigen NA14. The fusions co-localized with the wild-type proteins when expressed moderately. Overexpression of centrin-GFP and DIP13-GFP resulted in the formation of large aggregates and disturbed the distribution of the respective wild-type proteins. The amount of wild-type DIP13 was significantly reduced in cells overexpressing DIP13-GFP. Moreover, the cells frequently failed to assemble full-length flagella and flagellar regeneration was delayed, indicating a role of DIP13 during flagellar assembly. In contrast, overexpression of GFP-SFA, which retained more wild-type properties than SFA-GFP, increased the size of the striated fibers without altering the cross-shaped pattern. Abnormal patterns were observed in centrin-deficient cells, suggesting that centrin is required for proper localization of SFA. Photobleaching of GFP-SFA fibers indicated that GFP-SFA in the fibers is turned over slowly. Conditionally expressed centrin-GFP was incorporated into NBBCs in regions close to the basal bodies, but underrepresented in the dCF, indicative of a different dynamic of these two centrin fibers. Bending of the NBBCs was observed in vivo during flagellar motion, indicating that the filaments are flexible. In conclusion, in Chlamydomonas GFP-tagging is a useful tool for yielding new insights into the function and properties of the analyzed proteins.

Algal Proteins↗

PubMed2004

Isolation of highly pure and viable primordial germ cells from rainbow trout by GFP-dependent flow cytometry.

A highly pure and viable primordial germ cell (PGC) population appears to be an essential tool for establishing a cell line that can differentiate into a germ cell lineage and for studying the molecular biology and biochemistry of fish PGCs. Therefore, the aim of the present study was to establish a flow cytometric method for isolating highly pure and viable PGCs. As the material for PGC isolation, we used transgenic rainbow trout possessing the green fluorescent protein (GFP) gene driven by trout vasa-gene regulatory sequences (pvasa-GFP). Four independent transgenic strains were subjected to fluorescence microscopy and GFP-dependent flow cytometric analyses. We found that some of the pvasa-GFP transgenic strains exhibited ectopic background green fluorescence in the somatic cells aside from strong fluorescence in PGCs. Although flow cytometric analysis of genital ridge somatic cells in the four pvasa-GFP transgenic strains revealed a wide range of GFP intensities, we proved that somatic cell contamination of the GFP-positive cell population was markedly reduced if transgenic strains without the ectopic background green fluorescence were used. In addition, the forward light-scattering (FS) property, which is an indication of relative cell size, and the side light-scattering (SS) property, which is determined by cell shape and granularity, were employed to remove non-PGC contaminants from the GFP-positive cell population. By isolating GFP-positive cells with high FS/SS values, we were able to effectively remove cell blebs and the apoptotic fraction. Consequently, the purities and survival rates of isolated PGCs were greatly improved compared with those using GFP intensity as a single indicator. Thus, our flow cytometric method, in combination with the selection of suitable transgenic strains without the ectopic background green fluorescence, is capable of isolating highly pure and viable PGCs from rainbow trout. By using this method in combination with cell-cryopreservation and cell transplantation techniques, the isolated PGCs may also be used for preserving the genetic resources of endangered fish species and domesticated fish strains carrying commercially valuable traits. Mol. Reprod. Dev. 67: 91-100, 2004.

Animals↗

PubMed2001

Characterization of a metabotropic glutamate receptor type 5-green fluorescent protein chimera (mGluR5-GFP): pharmacology, surface expression, and differential effects of Homer-1a and Homer-1c.

Metabotropic glutamate receptor 5 (mGluR5) can modulate synaptic transmission by increasing intracellular Ca2+ and it plays a role in several forms of synaptic plasticity. We have constructed a fusion of human mGluR5 and green fluorescent protein (mGluR5-GFP). Expression of mGluR5-GFP in clonal cell lines yielded a functional fluorescent receptor with pharmacological profiles similar to wild-type mGluR5. mGluR5-GFP coimmunoprecipitated with Homer-1c, indicating that addition of GFP to the C-terminal did not prevent Homer binding. Coexpression of wild-type mGluR5 or mGluR5-GFP with Homer 1c, but not Homer-1a, resulted in reduced receptor surface localization and the formation of intracellular clusters. Neither Homer-1a nor Homer-1c had any effect on mGluR1 or mGluR1-GFP distribution. mGluR5-GFP expressed alone or in combination with Homer-1a formed dimers in HEK cells. Coexpression with Homer-1c, however, prevented mGluR5-GFP dimerization. Neither Homer altered the agonist profiles of mGluR5 or mGluR5-GFP. These data indicate that the functional expression of mGluR5 is regulated by Homer-1c and demonstrate that mGluR5-GFP provides a useful tool to study the molecular pharmacology and cell biology of mGluRs in real-time.

Animals↗

PubMed2005

Tracking nucleolar dynamics with GFP-Nopp140 during Drosophila oogenesis and embryogenesis.

We expressed two green fluorescent protein (GFP)-tagged Nopp140 isoforms in transgenic Drosophila melanogaster to study nucleolar dynamics during oogenesis and early embryogenesis. Specifically, we wanted to test whether the quiescent oocyte nucleus stored maternal Nopp140 and then to determine precisely when nucleoli formed during embryogenesis. During oogenesis nurse cell nucleoli accumulated GFP-Nopp140 gradually such that posterior nurse cell nucleoli in egg chambers at stage 10 were usually brighter than the more anterior nurse cell nucleoli. Nucleoli within apoptotic nurse cells disassembled in stages 12 and 13, but not all GFP-Nopp140 entered the oocyte through inter-connecting cytoplasmic bridges. Oocytes, on the other hand, lost their nucleoli by stage 3, but GFP-Nopp140 gradually accumulated in oocyte nuclei during stages 8-13. Most oocyte nuclei at stage 10 stored GFP-Nopp140 uniformly, but many stage 10 oocytes accumulated GFP-Nopp140 in presumed endobodies or in multiple smaller spheres. All oocyte nuclei at stages 11-12 were uniformly labeled, and GFP-Nopp140 diffused to the cytoplasm upon nuclear disassembly in stage 13. GFP-Nopp140 reappeared during embryogenesis; initial nucleologenesis occurred in peripheral somatic nuclei during embryonic stage 13, one stage earlier than reported previously. These GFP-Nopp140-containing foci disassembled at the 13th syncytial mitosis, and a second nucleologenesis occurred in early stage 14. The resulting nucleoli occupied nuclear regions closest to the periphery of the embryos. Pole cells contained GFP-Nopp140 during the syncytial embryonic stages, but their nucleologenesis started at gastrulation.

Animals↗

PubMed2006

GFP reporters detect the activation of the Drosophila JAK/STAT pathway in vivo.

JAK/STAT signaling is essential for a wide range of developmental processes in Drosophila melanogaster. The mechanism by which the JAK/STAT pathway contributes to these processes has been the subject of recent investigation. However, a reporter that reflects activity of the JAK/STAT pathway in all Drosophila tissues has not yet been developed. By placing a fragment of the Stat92E target gene Socs36E, which contains at least two putative Stat92E binding sites, upstream of GFP, we generated three constructs that can be used to monitor JAK/STAT pathway activity in vivo. These constructs differ by the number of Stat92E binding sites and the stability of GFP. The 2XSTAT92E-GFP and 10XSTAT92E-GFP constructs contain 2 and 10 Stat92E binding sites, respectively, driving expression of enhanced GFP, while 10XSTAT92E-DGFP drives expression of destabilized GFP. We show that these reporters are expressed in the embryo in an overlapping pattern with Stat92E protein and in tissues where JAK/STAT signaling is required. In addition, these reporters accurately reflect JAK/STAT pathway activity at larval stages, as their expression pattern overlaps that of the activating ligand unpaired in imaginal discs. Moreover, the STAT92E-GFP reporters are activated by ectopic JAK/STAT signaling. STAT92E-GFP fluorescence is increased in response to ectopic upd in the larval eye disc and mis-expression of the JAK kinase hopscotch in the adult fat body. Lastly, these reporters are specifically activated by Stat92E, as STAT92E-GFP reporter expression is lost cell-autonomously in stat92E homozygous mutant tissue. In sum, we have generated in vivo GFP reporters that accurately reflect JAK/STAT pathway activation in a variety of tissues. These reporters are valuable tools to further investigate and understand the role of JAK/STAT signaling in Drosophila.

Animals↗

PubMed2006

Purification of GFP fusion proteins from transgenic plant cell cultures.

Green fluorescence protein (GFP) has become a widely used reporter in many areas of life science. Monitoring foreign protein expression via GFP fusion is also very appealing for bioprocess applications. GFP itself has been purified from recombinant organisms by several methods, often involving unfavorable conditions (e.g., use of organic solvents and/or low pH) that may be destabilizing to some proteins. In this study, we have developed a general recovery scheme that entails a simple three-step purification procedure for GFP fusion proteins produced in tobacco suspension cells, with the intent of maximizing purity and yield under gentle conditions so as to maintain the integrity of the fusion partner. Ammonium sulfate treatment at 30% (v/v) precipitated particulate matter and removed aggregated material while simultaneously maintaining GFP solubility and increasing hydrophobicity. Hydrophobic interaction chromatography was then performed to eliminate the majority of background proteins while eluting GFP and fusions in a low ionic buffer suitable to be directly applied to an ion-exchange column as the final step. Three intracellular proteins, secreted alkaline phosphatase (SEAP), and granulocyte-macrophage colony-stimulating factor (GMCSF), each fused to GFP, as well as GFP itself, were recovered with yields exceeding 70% and purity levels over 80%. This purification scheme exploits the hydrophobic nature of GFP while maintaining a gentle environment for labile fusion partners. Although some optimization may be required, we believe this scheme may serve as a benchmark for purifying other GFP fusion proteins.

Alkaline Phosphatase↗

PubMed2001

Efficient GFP mutations profoundly affect mRNA transcription and translation rates.

Green fluorescent protein (GFP) variants with higher expression efficiencies have been generated by mutagenesis. Favorable mutations often improve the folding of GFP. However, an effect on protein folding fails to explain the efficiency of several other GFP mutations. In this work, we demonstrate that mutations of the GFP open reading frame and untranslated regions profoundly affect mRNA transcription and translation efficiencies. The removal of the GFP 5' untranslated region halves the transcription rate of the GFP gene, but hugely improves its translation rate. Mutations of the GFP open reading frame or the addition of peptide sequences differentially reduce the GFP mRNA transcription rate, translation efficiency and protein stability. These previously unrecognized effects are demonstrated to be critical to the efficiency of GFP mutants. These findings indicate the feasibility of generating more efficient GFP variants, with optimized mRNA transcription and translation in eukaryotic cells.

5' Untranslated Regions↗

PubMed1996

Double labelling of subcellular structures with organelle-targeted GFP mutants in vivo.

BACKGROUND: The green fluorescent protein (GFP) of Aequorea victoria is emerging as a unique tool for monitoring complex phenomena such as gene expression and organelle structure and dynamics in living cells. The recent description of GFP mutants with modified spectral properties opens numerous new applications in cell biology. However, the expression and the characteristics of these GFP mutants in living eukaryotic cells have not been verified yet. RESULTS: Here, we demonstrate the usefulness of the GFP mutants for cell biology studies in vivo, by the use of wild-type GFP, a 'bright' GFP mutant (S65T) and a mutant with blue-shifted excitation and emission spectra (Y66H/Y145F). We have constructed two GFP chimeras targeted to mitochondria, mtGFP(S65T) and mtGFP(Y66H/Y145F), with the same strategy used previously for mtGFP. In addition, two GFP chimeras targeted to the nucleus, nuGFP and nuGFP(S65T), were constructed by fusing the wild-type GFP or the (S65T) mutant to the rat glucocorticoid receptor. By co-transfecting mtGFP(Y66H/Y145F) and nuGFP, the nucleus and the mitochondria were visualized simultaneously in living cells. Similarly, mtGFP and mtGFP(Y66H/Y145F) were transfected into different populations of cells, and the events of cellular fusion, and mitochondrial intermixing and/or fusion, were directly monitored. CONCLUSIONS: The successful expression of organelle-targeted GFP mutants in live eukaryotes expands the uses of this fluorescent protein in cell biology, allowing direct access to key biological issues, such as the study of the interactions of different organelles in vivo. These results also open the way to other exciting applications, such as the direct study of protein redistribution and protein-protein interactions in living cells.

Base Sequence↗