Rapid protein-folding assay using green fluorescent protein.

Formation of the chromophore of green fluorescent protein (GFP) depends on the correct folding of the protein. We constructed a "folding reporter" vector, in which a test protein is expressed as an N-terminal fusion with GFP. Using a test panel of 20 proteins, we demonstrated that the fluorescence of Escherichia coli cells expressing such GFP fusions is related to the productive folding of the upstream protein domains expressed alone. We used this fluorescent indicator of protein folding to evolve proteins that are normally prone to aggregation during expression in E. coli into closely related proteins that fold robustly and are fully soluble and functional. This approach to improving protein folding does not require functional assays for the protein of interest and provides a simple route to improving protein folding and expression by directed evolution.     

Establishment of insect cell lines expressing green fluorescent protein on cell surface based on AcMNPV GP64 membrane fusion characteristic

Displaying a protein on the surface of cells has been provided a very successful strategy to function research of exogenous proteins. Based on the membrane fusion characteristic of Autographa californica multiple nucleopolyhedrovirus envelope protein GP64, we amplified and cloned N-terminal signal peptide and C-terminal transmembrane domain as well as cytoplasmic tail domain of gp64 gene into vector pIZ/V5-His with multi-cloning sites to construct the cell surface expression vector pIZ/V5-gp64. To verify that the vector can be used to express proteins on the membrane of insect cells, a recombinant plasmid pIZ/V5-gp64-GFP was constructed by introducing the PCR amplified green fluorescent protein (GFP) gene and transfected into insect cell lines Sf9 and H5. The transected cells were screened with zeocin and cell cloning. PCR verification results showed that the GFP gene was successfully integrated into these cells. Green fluorescence in Sf9-GFP and H5-GFP cells was observed by using confocal laser scanning microscopy and immunofluorescence detection indicated that GFP protein was located on the cell membrane. Western blot results showed that a fusion protein GP64-GFP of about 40 kDa was expressed on the membrane of Sf9-GFP and H5-GFP cells. The expression system constructed in this paper can be used for localization and continuous expression of exogenous proteins on insect cell membrane.     

Rapid establishment of high-producing cell lines using dicistronic vectors with glutamine synthetase as the selection marker

Recombinant proteins are useful tools in biological research, drug development, and drug screening. Specially designed expression vectors have been developed to introduce cDNA for recombinant protein expression in mammalian cells. We have combined a discistronic mRNA design for expression of the recombinant protein, using glutamine synthetase (GS) for selection. A soluble form of human interleukin-4 receptor alpha chain was used as the model protein. The dicistronic vectors were compared to a standard expression vector in CHO-K1 cells in parallel experiments. Our data showed that a dicistronic vector containing an internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV) was superior to a conventional expression vector in both levels of protein expression and amplification efficiency. The productivity of these clones was stable without selection pressure for an extended period of time. The GS selection system within a dicistronic vector design can achieve rapid and efficient gene amplification for protein production.     

Integrated bioprocessing in Saccharomyces cerevisiae using green fluorescent protein as a fusion partner

In this study, we examine the use of green fluorescent protein (GFP) for monitoring a hexokinase (HXK)-GFP fusion protein in Saccharomyces cerevisiae for various events including expression, degradation, purification, and localization. The fusion, HXK-EK-GFP-6 x His, was constructed where the histidine tag (6 x His) would allow for convenient affinity purification, and the enterokinase (EK) cleavage site would be used for separation of HXK from GFP after affinity purification. Our results showed that both HXK and GFP remained active in the fusion and, more importantly, that there was a linear correlation between HXK activity and GFP fluorescence. Enterokinase cleavage studies revealed that both GFP fluorescence intensity and HXK activity remained unchanged after separation of the fusion proteins, which indicated that fusion of GFP did not cause structural alteration of HXK and thus did not affect the enzymatic activity of HXK. We also found that degradation of the fusion protein occurred, and that degradation was limited to HXK with GFP remaining intact in the fusion. Confocal microscopy studies showed that while GFP was distributed evenly in the yeast cytosol, HXK-GFP fusion followed the correct localization of HXK, which resulted in a di-localization of both cytosol and the nucleus. GFP proved to be a useful fusion partner that may lead to the possibility of integrating the bioprocesses by quantitatively following the entire process visually.     

A flexible single-step detection of blotted antigen using a fusion protein between protein A and green fluorescent protein

A green fluorescent protein mutant (S147P GFP) was fused with protein A and expressed in Escherichia coli. This fusion protein (PA-GFP147) was used in immunoblotting studies as a new detection system, designated as "flexible single-step detection (FSSD)". In FSSD, the detection of blotted antigen was done in one step, and the antigen-antibody reaction can be monitored by UV-irradiation in real time. The reaction time, therefore, can be flexibly controlled by monitoring the green fluorescence.     

Rapid production of a chimeric antibody-antigen fusion protein based on 2A-peptide cleavage and green fluorescent protein expression in CHO cells

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.     

Rapid and efficient selection of yeast displaying a target protein using thermo-responsive magnetic nanoparticles

Magnetic separation provides a relatively quick and easy-to-use method for cell isolation and protein purification. We have developed a rapid and efficient procedure to isolate yeast cells displaying a target polypeptide, namely, the Staphylococcus aureus ZZ domain, which serves as s model for protein interactions and can bind immunoglobulin G (IgG). We optimized selection of ZZ-displaying yeast cells using thermoresponsive magnetic nanoparticles. A model library was prepared by mixing various proportions of target yeast displaying the ZZ domain with control cells. Target cells in the model library that bound to the ZZ-specific binding partner, biotinylated IgG, were selected with biotinylated thermoresponsive magnetic nanoparticles using the biotin-avidin sandwich system. We determined ZZ expression levels and optimized the concentrations of both magnetic nanoparticles and avidin for efficient selection of target cells. After optimization, we successfully enriched the target cell population 4700-fold in a single round of selection. Moreover, only two rounds of selection were required to enrich the target cell population from 0.001% to nearly 100%. Our results suggest that magnetic separation will be useful for efficient exploration of novel protein-protein interactions and rapid isolation of biomolecules with novel functions.     

Amino terminal interaction in the prion protein identified using fusion to green fluorescent protein

In contrast to the well-characterized carboxyl domain, the amino terminal half of the mature cellular prion protein has no defined structure. Here, following fusion of mouse prion protein fragments to green fluorescence protein as a reporter of protein stability, we report extreme variability in fluorescence level that is dependent on the prion fragment expressed. In particular, exposure of the extreme amino terminus in the context of a truncated prion protein molecule led to rapid degradation, whereas the loss of only six amino terminal residues rescued high level fluorescence. Study of the precise endpoints and residue identity associated with high fluorescence suggested a domain within the amino terminal half of the molecule defined by a long-range intramolecular interaction between 23KKRPKP28 and 143DWED146 and dependent upon the anti-parallel beta-sheet ending at residue 169 and normally associated with the structurally defined carboxyl terminal domain. This previously unreported interaction may be significant for understanding prion bioactivity and for structural studies aimed at the complete prion structure.     

Rapid screening of potential autophagic inductor agents using mammalian cell lines

Recent progress in understanding the molecular basis of autophagy has demonstrated its importance in several areas of human health. Affordable screening techniques with higher sensitivity and specificity to identify autophagy are, however, needed to move the field forward. In fact, only laborious and/or expensive methodologies such as electron microscopy, dye-staining of autophagic vesicles, and LC3-II immunoblotting or immunoassaying are available for autophagy identification. Aiming to fulfill this technical gap, we describe here the association of three widely used assays to determine cell viability – Crystal Violet staining (CVS), 3-[4, 5-dimethylthiaolyl]-2, 5-diphenyl-tetrazolium bromide (MTT) reduction, and neutral red uptake (NRU) – to predict autophagic cell death in vitro. The conceptual framework of the method is the superior uptake of NR in cells engaging in autophagy. NRU was then weighted by the average of MTT reduction and CVS allowing the calculation of autophagic arbitrary units (AAU), a numeric variable that correlated specifically with the autophagic cell death. The proposed strategy is very useful for drug discovery, allowing the investigation of potential autophagic inductor agents through a rapid screening using mammalian cell lines B16-F10, HaCaT, HeLa, MES-SA, and MES-SA/Dx5 in a unique single microplate.     

Establishment of porcine transgenic embryonic germ cell lines expressing enhanced green fluorescent protein

The purpose of this study was to isolate porcine embryonic germ (EG) cells and establish transgenic EG cell lines. Plasmid DNA was the enhanced green fluorescent protein (EGFP) vector. Porcine EG cells in rapid proliferation (4th to 9th passage) were transfected with LipofecTamine 2000 and TransFast reagents. Porcine EG cells transfected with a complex of 1 microg of DNA and 2 microL of LipofecTamine 2000 reagent yielded four EG-EGFP cell lines, which emitted bright green fluorescence. EG-EGFP cells cultured for more than 2 weeks without passage gave rise to various differentiated phenotypes. In addition, to determine the degree to which EG cells become integrated into the inner cell mass of host embryos, 135 embryos were injected with porcine EG-EGFP cells; 110 embryos survived and developed into blastocysts (81.5%). Eighty-four chimeric embryos contained fluorescent cells after culture; 49 blastocysts contained EG-EGFP cells in the inner cell mass. Our results suggested that the chimeric rate would not be improved via using different stages of embryos for injection.     

Differential localization of protein kinase C delta by phorbol esters and related compounds using a fusion protein with green fluorescent protein

Enzyme localization often plays a controlling role in determining its activity and specificity. Protein kinase C (PKC) has long been known to translocate in response to physiological stimuli as well as to exogenous ligands such as the phorbol esters. We report here that different phorbol derivatives and related ligands, selected for differences in chemical structure and profile of biological activity, induce distinct patterns of redistribution of PKC delta. Localization of a PKC delta-green fluorescent protein (GFP) fusion construct was monitored in living Chinese hamster ovary cells as a function of ligand, concentration, and time using confocal laser scanning microscopy. delta-PKC-GFP was expressed predominantly in the cytoplasm, with some in the nucleus and perinuclear region. Phorbol 12-myristate 13-acetate (PMA) induced plasma membrane translocation followed by slower nuclear membrane translocation. As the concentration of PMA increased, the proportion of nuclear to plasma membrane localization increased markedly. In contrast to PMA, bryostatin 1, a unique activator of PKC that induces a subset of PMA-mediated responses while antagonizing others, at all doses induced almost exclusively nuclear membrane translocation. Like PMA, the complete tumor promoter 12-deoxyphorbol 13-tetradecanoate induced plasma membrane and slower nuclear membrane translocation, whereas the inhibitor of tumor promotion 12-deoxyphorbol 13-phenylacetate, which differs only in its side chain, induced a distinctive distribution of PKC delta-GFP. Finally, the novel constrained diacylglycerol derivative B8-DL-B8 induced a slow Golgi localization. We speculate that differential control of PKC delta localization may provide an interesting strategy for producing ligands with differential biological consequences.     

Assessment of hypoxia inducible factor levels in cancer cell lines upon hypoxic induction using a novel reporter construct

Hypoxia Inducible Factor (HIF) signaling pathway is important for tumor cells with limited oxygen supplies, as it is shown to be involved in the process of proliferation and angiogenesis. Given its pivotal role in cancer biology, robust assays for tracking changes in HIF expression are necessary for understanding its regulation in cancer as well as developing therapies that target HIF signaling. Here we report a novel HIF reporter construct containing tandem repeats of minimum HIF binding sites upstream of eYFP coding sequence. We show that the reporter construct has an excellent signal to background ratio and the reporter activity is HIF dependent and directly correlates with HIF protein levels. By utilizing this new construct, we assayed HIF activity levels in different cancer cell lines cultured in various degrees of hypoxia. This analysis reveals a surprising cancer cell line specific variation of HIF activity in the same level of hypoxia. We further show that in two cervical cancer cell lines, ME180 and HeLa, the different HIF activity levels observed correlate with the levels of hsp90, a cofactor that protects HIF against VHL-independent degradation. This novel HIF reporter construct serves as a tool to rapidly define HIF activity levels and therefore the therapeutic capacity of potential HIF repressors in individual cancers.     

High-throughput clonal selection of recombinant CHO cells using a dominant selectable and amplifiable metallothionein-GFP fusion protein

Transfected mammalian cells can be used for the production of fully processed recombinant proteins for medical and industrial purposes. However, the isolation of high-producing clones is traditionally time-consuming. Therefore, we developed a high-throughput screening method to reduce the time and effort required to isolate high-producing cells. This involved the construction of an expression vector containing the amplifiable gene metallothionein (MT), fused in-frame to green fluorescent protein (GFP). The fusion gene (MTGFP) confers metal resistance similar to that of the wild-type metallothionein and expression can be monitored using either flow cytometry or a fluorometer to measure green fluorescence. Expression of MTGFP acted as a dominant selectable marker allowing rapid and more efficient selection of clones at defined metal concentrations than with the antibiotic G418. Cells harboring MTGFP responded to increasing metal concentrations with a corresponding increase in fluorescence. There was also a corresponding increase in recombinant protein production, indicating that MTGFP could be used as a selectable and amplifiable gene for the coexpression of foreign genes. Using our expression vector encoding MTGFP, we demonstrate a high-throughput clonal selection protocol for the rapid isolation of high-producing clones from transfected CHO cells. We were able to isolate cell lines reaching specific productivities of >10 microg hGH/10(6) cells/day within 4 weeks of transfection. The advantage of this method is that it can be easily adapted for automated procedures using robotic handling systems.     

In vivo study of trichoderma-pathogen-plant interactions, using constitutive and inducible green fluorescent protein reporter systems

Plant tissue colonization by Trichoderma atroviride plays a critical role in the reduction of diseases caused by phytopathogenic fungi, but this process has not been thoroughly studied in situ. We monitored in situ interactions between gfp-tagged biocontrol strains of T. atroviride and soilborne plant pathogens that were grown in cocultures and on cucumber seeds by confocal scanning laser microscopy and fluorescence stereomicroscopy. Spores of T. atroviride adhered to Pythium ultimum mycelia in coculture experiments. In mycoparasitic interactions of T. atroviride with P. ultimum or Rhizoctonia solani, the mycoparasitic hyphae grew alongside the pathogen mycelia, and this was followed by coiling and formation of specialized structures similar to hooks, appressoria, and papillae. The morphological changes observed depended on the pathogen tested. Branching of T. atroviride mycelium appeared to be an active response to the presence of the pathogenic host. Mycoparasitism of P. ultimum by T. atroviride occurred on cucumber seed surfaces while the seeds were germinating. The interaction of these fungi on the cucumber seeds was similar to the interaction observed in coculture experiments. Green fluorescent protein expression under the control of host-inducible promoters was also studied. The induction of specific Trichoderma genes was monitored visually in cocultures, on plant surfaces, and in soil in the presence of colloidal chitin or Rhizoctonia by confocal microscopy and fluorescence stereomicroscopy. These tools allowed initiation of the mycoparasitic gene expression cascade to be monitored in vivo.     

Functional analysis of type 1alpha cGMP-dependent protein kinase using green fluorescent fusion proteins

The cGMP-dependent protein kinases (PKGs) are ubiquitous effector enzymes that regulate a variety of physiological processes in response to nitric oxide and natriuretic agonists. We have constructed green fluorescent fusion proteins (GFP) using full-length (PKG-GFP) and truncations encoding either the regulatory domain of PKG1alpha (G1alphaR-GFP) or the catalytic domains of PKG1alpha (GFP-G1C) to examine the enzymatic properties and intracellular location. When transiently transfected into mammalian cells, these constructs were detected on Western blots at the expected sizes using anti-GFP antibodies. The GFP-G1C and the full-length PKG1alpha-GFP fusion proteins were found to have constitutive activity both in vivo and in vitro. The G1alphaR-GFP protein was found to dimerize with endogenous type 1 PKG and behaved in a dominant negative manner both in vivo and in vitro. When expressed transiently in either HEK-293 or A549 epithelial cells, the fusion proteins encoding the amino-terminal regulatory domains (PKG-GFP, G1alphaR-GFP) were present in the cytosol and were rarely observed in the nucleus. In contrast, the GFP-G1C (lacking regulatory domains) concentrated in the nucleus. Of the fusion proteins containing the regulatory region, the constitutive PKG-GFP protein was present in a more centralized location, whereas the G1alphaR-GFP protein colocalized with F-actin on stress fibers and in dynamic regions of the plasma membrane. Microscopic and immunoprecipitation studies indicated that both the G1alphaR-GFP and the PKG-GFP fusion proteins colocalized with vasodilator-stimulated phosphoprotein (VASP). These constructs thus represent novel tools with which to visualize inactive, and activated, PKG1alpha in vivo, and we have used them to demonstrate two functionally independent domains. In addition, we show for the first time in living cells that PKG is found in dynamic membrane regions in association with VASP.     

Calmodulin-binding protein detection using a non-radiolabeled calmodulin fusion protein

Calmodulin-binding proteins are involved in numerous cellular signaling pathways. The biotinylated-calmodulin overlay is a nonradioactive method widely used to detect calmodulin-binding proteins in tissue and cell samples. This method has several limitations; therefore, we developed a nonradioactive calmodulin-binding protein detection overlay using an S-tag-labeled calmodulin fusion protein. An expression system was used to generate a calmodulin fusion protein with an S-tag label, a 15 amino acid sequence that binds to a 105 amino acid S-protein. The S-protein is conjugated to horseradish peroxidase for final detection with a chemiluminescent substrate. The S-tag calmodulin was compared to purified calmodulin and biotinylated calmodulin in a calmodulin-dependent phosphodiesterase assay. The results of the calmodulin-dependent phosphodiesterase assay indicate that S-tag calmodulin induces higher phosphodiesterase activity than biotinylated calmodulin and lower activity than purified calmodulin. A comparison of the biotinylated and S-tag calmodulin overlay assays indicate that S-tag calmodulin is more sensitive than biotinylated calmodulin in the detection of calcineurin, a known calmodulin-binding protein. The overlay assay results also indicate that the S-tag calmodulin and biotinylated calmodulin detect similar calmodulin-binding proteins in colon epithelial cells. In conclusion, the S-tag calmodulin overlay assay is a consistent, sensitive, and rapid nonradioactive method to detect calmodulin-binding proteins.     

Rapid obtention of stable,bioluminescent tumor cell lines using a tCD2-luciferase chimeric construct

Background  

Bioluminescent tumor cell lines are experimental tools of major importance for cancer investigation, especially imaging of tumors in xenografted animals. Stable expression of exogenous luciferase in tumor cells combined to systemic injection of luciferin provides an excellent signal/background ratio for external optical imaging. Therefore, there is a need to rationalize and speed up the production of luciferase-positive tumor cell lines representative of multiple tumor phenotypes. For this aim we have designed a fusion gene linking the luciferase 2 protein to the c-terminus of a truncated form of the rat CD2 protein (tCD2-luc2). To allow simultaneous assessment of the wild-type luciferase 2 in a context of tCD2 co-expression, we have made a bicistronic construct for concomitant but separate expression of these two proteins (luc2-IRES-tCD2). Both the mono- and bi-cistronic constructs were transduced in lymphoid and epithelial cells using lentiviral vectors.     

Production of recombinant polyhedra containing Cry1Ac fusion protein in insect cell lines

Insect cell lines and the control of infection for obtaining the maximum amount of polyhedrin-CrylAc-polyhedrin fusion protein from Bactrus in monolayer and suspension culture systems were tested. Growth rates of the Trichoplusia ni (High-Five) cell line in both culture systems were better than the other insect cell lines, Spodoptera fiugiferda (Sf-9, Sf-21), Trichoplusia ni (Tn5), and Spodoptera exigua (Se301). The expression of the fusion protein in a monolayer culture showed that Se301 cells were 2.3-4.8 times more productive on a per cell basis than the other cell lines. However, in suspension culture, only High-Five cells were productive. High-Five cells infected with Bactrus at a multiplicity of infection (MOI) of 5 and a cell density of 3.0 x 10(5) cells per ml were more productive than the other infection condition in a suspension culture suitable for a large-scale production of baculovirus. In conclusion, for the large-scale production of Bactrus in vitro, High-Five cells showing good growth and high productivity are suitable.     

Rapid titer assay of adenovirus containing green fluorescent protein gene using flow cytometric analysis

The plaque assay has been widely used for titration of adenovirus (AdV). However, it takes usually 2-3 weeks, so this slow assay often impedes bioprocess development of large-scale AdV production. In this study, we developed a rapid AdV titration assay that can be done within a day. Further, unlike the plaque assay, this assay does not require a laborious serial dilution of samples. This rapid assay can be achieved by using green fluorescent protein (GFP) as a marker gene and flow cytometric analysis. It yields a good correlation between infectious titer of AdV harboring GFP and flow cytometric parameters such as average green fluorescence intensity or % of infected cells. Taken together, this rapid assay will facilitate bioprocess development for efficient large-scale AdV production.