蛋白酪氨酸磷酸酶SHP-2在乳腺癌细胞移动及粘附中的作用

探讨蛋白酪氨酸磷酸酶SHP 2在乳腺癌细胞MCF 7的移动及粘附中的作用 .利用基因重组技术分别将野生型SHP 2与突变型SHP 2与绿色荧光蛋白GFP的基因片段构成重组质粒 (SHP 2 GFP、SHP 2C >S GFP) .脂质体转染法分别转入MCF 7中 ,表达成功后筛选并建立SHP 2 GFP和SHP 2C >S GFP细胞株 .荧光显微镜观察细胞移动情况 ,免疫印迹法检测粘附分子E 钙粘蛋白和金属蛋白酶MMP 1及MMP 9的表达 .实验后建立SHP 2 GFP及SHP 2C >S GFP细胞株 ,同时观察到SHP 2C >S GFP细胞的形态发生明显改变 :从梭形状态变成圆形状态 .荧光显微镜发现 ,MCF 7细胞和SHP 2 GFP、SHP 2C >S GFP转染的细胞在 3h、6h、9h的移动情况分别是MCF 7为 10 %、2 3%、5 4% ,SHP 2 GFP为 15 %、4 9%、98% ,SHP 2C >S GFP为 4 %、11%、30 % .免疫印迹结果表明 ,SHP 2C >S GFP细胞的E 钙粘蛋白表达比SHP 2 GFP细胞明显升高 (P <0 0 5 ) .MMP 1及MMP 9的表达量在SHP 2 GFP细胞中有所增强 (P <0 0 5 ) .实验表明 ,SHP 2可能通过调节粘附分子和基质金属磷酸酶而在细胞移动、粘附中发挥重要作用     

Engineering green fluorescent protein as a dual functional tag

A hexa-histidine (6 x His) sequence was inserted into a surface loop of the green fluorescent protein (GFP) to develop a dual functional GFP useful for both monitoring and purification of recombinant proteins. Two variants (GFP172 and GFP157), differentiated by the site of insertion of the 6xHis sequence, were developed and compared with a control variant (GFPHis) having the 6xHis sequence at its C-terminus. The variants were produced in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC). The purification efficiencies by IMAC for all variants were found to be comparable. Purified GFP172 and GFP157 variants retained approximately 60% of the fluorescence compared to that of GFPHis. The reduction in the fluorescence intensity associated with GFP172 and GFP157 was attributed to the lower percentage of fluorescent GFP molecules in these variants. Nonetheless, the rates of fluorescence acquisition were found to be similar for all functional variants. Protein misfolding at an elevated temperature (37 degrees C) was found to be less profound for GFP172 than for GFP157. The dual functional properties of GFP172 were tested with maltose binding protein (MBP) as the fusion partner. The MBP-GFP172 fusion protein remained fluorescent and was purified from E. coli lysate as well as from spiked tobacco leaf extracts in a single-step IMAC. For the latter, a recovery yield of approximately 75% was achieved and MBP-GFP172 was found to coelute with a degraded product of the fusion protein at a ratio of about 4:1. The primary advantage of the chimeric GFP tag having an internal hexa-histidine sequence is that such a tag allows maximum flexibility for protein or peptide fusions since both N- and C-terminal ends of the GFP are available for fusion.     

Purification of GFP fusion proteins with high purity and yield by monoclonal antibody-coupled affinity column chromatography

GFP has often been used as a marker of gene expression, protein localization in living and fixed tissues as well as for protein targeting in intact cells and organisms. Monitoring foreign protein expression via GFP fusion is also very appealing for bioprocess applications. Many cells, including bacterial, fungal, plant, insect and mammalian cells, can express recombinant GFP (rGFP) efficiently. Several methods and procedures have been developed to purify the rGFP or recombinant proteins fused with GFP tag. However, most current GFP purification methods are limited by poor yields and low purity. In the current study, we developed an improved purification method, utilizing a FMU-GFP.5 monoclonal antibody (mAb) to GFP together with a mAb-coupled affinity chromatography column. The method resulted in a sample that was highly pure (more than 97% homogeneity) and had a sample yield of about 90%. Moreover, the GFP epitope permitted the isolation of almost all the active recombinant target proteins fused with GFP, directly and easily, from the crude cellular sources. Our data suggests this method is more efficient than any currently available method for purification of GFP protein.     

Purification of recombinant GFP produced by Agrobacterium-mediated transient expression in Nicotiana excelsior

Green fluorescent protein (GFP) is commonly used as a reporter protein in a wide range of biological experiments. The efficient protocol of Agrobacterium-mediated transient expression in Nicotiana excelsior was applied for quick preparative production of recombinant GFP. The protein purification scheme has been developed and includes ammonium sulfate precipitation and Q-sepharose anion-exchange chromatography. It results in the obtaining of a fraction with about 85% GFP homogeneity and the protein yield of about 75%. The text was submitted by the authors in English.     

High expression of green fluorescent protein in Pichia pastoris leads to formation of fluorescent particles

Wild type gene for green fluorescent protein (GFP) was stably integrated into the Pichia pastoris genome and yielded an expression level of over 40% of total cellular protein. The high cytoplasmic concentration of fluorescent (properly folded and processed) GFP caused the formation of fluorescent spherical structures, which could be observed by fluorescence or confocal microscopy after controlled permeabilization of the yeast cells with 0.2% N-lauroyl sarcosine (NLS). Fluorescent GFP particles were also isolated after removal of the cell wall and found to be quite resistant to 0.2% N-lauroyl sarcosine. SDS-PAGE analysis of the isolated fluorescent particles revealed the presence of an 80 kDa protein (alcohol oxidase) and GFP (30%). We conclude that GFP is able to enter spontaneously into the peroxisomes and is inserted into densely packed layers of alcohol oxidase. Consequently, the formation of similar fluorescent particles can also be expected in other organisms when using high-level expression systems. As GFP is widely used in fusion with other proteins as a reporter for protein localization and for many other applications in biotechnology, care must be taken to avoid false interpretations of targeting or trafficking mechanisms inside the cells. In addition, when whole cells or cytoplasmic fractions are used for the quantitative determination of GFP levels, incorrect and misleading values of GFP could be obtained due to the formation of fluorescent particles containing material inside which is not available for fluorescence measurements.     

Non-targeted and targeted protein movement through plasmodesmata in leaves in different developmental and physiological states

Plant cells rely on plasmodesmata for intercellular transport of small signaling molecules as well as larger informational macromolecules such as proteins. A green fluorescent protein (GFP) reporter and low-pressure microprojectile bombardment were used to quantify the degree of symplastic continuity between cells of the leaf at different developmental stages and under different growth conditions. Plasmodesmata were observed to be closed to the transport of GFP or dilated to allow the traffic of GFP. In sink leaves, between 34% and 67% of the cells transport GFP (27 kD), and between 30% and 46% of the cells transport double GFP (54 kD). In leaves in transition transport was reduced; between 21% and 46% and between 2% and 9% of cells transport single and double GFP, respectively. Thus, leaf age dramatically affects the ability of cells to exchange proteins nonselectively. Further, the number of cells allowing GFP or double GFP movement was sensitive to growth conditions because greenhouse-grown plants exhibited higher diffusion rates than culture-grown plants. These studies reveal that leaf cell plasmodesmata are dynamic and do not have a set size exclusion limit. We also examined targeted movement of the movement protein of tobacco mosaic virus fused to GFP, P30::GFP. This 58-kD fusion protein localizes to plasmodesmata, consistently transits from up to 78% of transfected cells, and was not sensitive to developmental age or growth conditions. The relative number of cells containing dilated plasmodesmata varies between different species of tobacco, with Nicotiana clevelandii exhibiting greater diffusion of proteins than Nicotiana tabacum.     

High-level secretion of functional green fluorescent protein from transgenic tobacco cell cultures: characterization and sensing

Green fluorescent protein (GFP) is useful for studying protein trafficking in plant cells. This utility could potentially be extended to develop an efficient secretory reporter system or to enable on-line monitoring of secretory recombinant protein production in plant cell cultures. Toward this end, the aim of the present study was to: (1) demonstrate and characterize high levels of secretion of fluorescent GFP from transgenic plant cell culture; and (2) examine the utility of GFP fluorescence for monitoring secreted recombinant protein production. In this study we expressed in tobacco cell cultures a secretory GFP construct made by splicing an Arabidopsis basic chitinase signal sequence to GFP. Typical extracellular GFP accumulation was 12 mg/L after 10 to 12 days of culture. The secreted GFP is functional and it accounts for up to 55% of the total GFP expressed. Findings from culture treatments with brefeldin A suggest that GFP is secreted by the cultured tobacco cells via the classical endoplasmic reticulum-Golgi pathway. Over the course of flask cultures, medium fluorescence increased with the secreted GFP concentrations that were determined using either Western blot or enzyme-linked immunoassay. Real-time monitoring of secreted GFP in plant cell cultures by on-line fluorescence detection was verified in bioreactor cultures in which the on-line culture fluorescence signals showed a linear dependency on the secreted GFP concentrations.     

Plasmodesmata transport of GFP alone or fused to potato virus X TGBp1 is diffusion driven

Summary. Plasmodesmata (Pd) provide a pathway for exchanging various macromolecules between neighboring plant cells. Researchers routinely characterize the mobility of the green-fluorescent protein (GFP) and GFP fusions through Pd by calculating the proportion of sites in bombarded leaves which show fluorescence in multiple cell clusters (% movement). Here, the Arrhenius equation was used to describe the temperature dependence of GFP and GFP-TGBp1 (potato virus X triple gene block protein1) movement, using % movement values, and to calculate the activation energy for protein transport. The resulting low activation energy indicates GFP and GFP-TGBp1 movement are diffusion driven. Furthermore, GFP movement is inversely proportional to the leaf surface area of expanding leaves. The increase in leaf area results mainly from cell expansion during the sink–source transition. The increasing cell size results in lower Pd density, which decreases the probability that a GFP attains an open Pd by diffusion. The decline in GFP movement as leaf area expands indicates that, in addition to GFP diffusion through Pd, attaining an open Pd by undirected diffusion might be limiting for Pd transport. In summary, this report provides a new quantitative method for studying Pd conductivity. Correspondence: Jeanmarie Verchot Lubicz, Department of Entomology and Plant Pathology, 127 Noble Research Center, Oklahoma State University, Stillwater, OK 74078, U.S.A.     

Green fluorescent protein as a secretory reporter and a tool for process optimization in transgenic plant cell cultures

Green fluorescent protein (GFP) is an attractive reporter for bioprocess monitoring. Although expression of GFP in plants has been widely reported, research on the use of GFP in plant cell cultures for bioprocess applications has been limited. In this study, the suitability of GFP as a secretory reporter and a useful tool in plant cell bioprocess optimization was demonstrated. GFP was produced and secreted from suspension cells derived from tobacco that was transformed with a binary vector containing mgfp5-ER cDNA, a modified GFP for efficient sorting to the endoplasmic reticulum, under control of the CaMV 35S promoter. For cell line gfp-13, extracellular and intracellular GFP accumulated to 15.4 and 29.4 mg x 1(-1), respectively. Extracellular GFP accounted for 30.9% of the total extracellular protein. The molecular mass of extracellular GFP was nearly identical to that of a recombinant GFP standard, indicating cleavage of the signal sequence. Neomycin phosphotransferase II, a cytosolic selection marker, was found almost exclusively in cellular extracts with less than 2% in the extracellular medium. These results suggest that extracellular GFP is most likely the result of secretion rather than nonspecific leakage from cells. Furthermore, medium fluorescence intensity correlated nicely with extracellular GFP concentration supporting the use of GFP as a quantitative secretory reporter. During the batch cultivation, culture GFP fluorescence also followed closely with cell growth. A medium feeding strategy was then developed based on culture GFP fluorescence that resulted in improved biomass as well as GFP production in a fed-batch culture.     

Use of the green fluorescent protein variant (YFP) to monitor MetArg human proinsulin production in Escherichia coli.

Green fluorescent protein (GFP), a relatively new reporter gene, is making an impact on many aspects of science. The attributes of GFP could also be applied to the area of recombinant protein production. The work described here represents the first experiments using GFP as a tool to monitor recombinant protein production in real time in the fermentation process. We have constructed plasmids containing an operon fusion of the gene encoding MetArg-human proinsulin and reporter gene GFP (GFP, BFP, and YFP variants). The MetArg-proinsulin and GFP variant reporter protein were overexpressed in Escherichia coli BL21(DE3) after isopropyl beta-d-thiogalactoside induction. The MetArg-proinsulin to YFP protein ratio did not change in the cells during the bioprocess. Since there is a quantitative relationship between the level of MetArg-proinsulin concentration and YFP fluorescence, it is possible to measure only YFP fluorescence in order to monitor the production of MetArg-proinsulin during the bioprocess. The expression level of MetArg-proinsulin could reach 20-25%. Some 140 mg recombinant MetArg-human proinsulin could be obtained easily from 1 liter of fermentation medium. The MetArg-proinsulin could simply be changed into human insulin by trypsin and carboxypeptidase B treatment in later steps. These experiments provide possibilities for using the YFP reporter gene as a convenient tool to monitor protein expression in biotechnological processes. The proposed technique could reduce the time- and labor-intensive analysis of protein production and would improve the efficiency of process development.     

Optimization of the green fluorescent protein (GFP) expression from a lactose-inducible promoter in Lactobacillus casei

An expression vector for Lactobacillus casei has been constructed containing the inducible lac promoter and the gene encoding ultraviolet visible green fluorescent protein (GFP(UV)) as reporter. Different conditions to grow L. casei were assayed and fluorescence as well as total protein synthesized were quantified. The maintenance of neutral pH had the greatest incidence on GFP(UV) expression, followed by aeration and a temperature of 30 degrees C. Environmental factors favoring GFP(UV) accumulation did not exactly correlate with those enhancing fluorescence. Therefore, oxygenation, by stirring the culture, had the greatest influence on the proportion of fluorescent protein, which is in accordance with the structural requirements of this protein. The highest yield obtained was 1.3 microg of GFP per mg of total protein, from which 55% was fluorescent.     

Silencing of Bcl-XL Expression in Human MGC-803 Gastric Cancer Cells by siRNA

To investigate the inhibitory effect of the Bcl-XL small interfering RNA(siRNA)on BcI-XLgene expression in the human gastric cancer cell line MGC-803,green fluorescent protein(GFP)siRNAwas constructed and transfected into MGC-803 ceils,together with GFP expression vector pTrace SV40.GFP expression levels were observed using fluorescence microscopy.Bcl-XL siRNA and negative siRNAwere then constructed and stably transfected into MGC-803 cells.RT-PCR and immunofluorescence wereused to detect the expression of Bcl-XL.Spontaneous apoptosis was detected by acridine orange(AO)andflow cytometry.Results were as follows:(1)48 h after GFP expression vector and GFP siRNA co-transfection,the expression level of GFP in the GFP siRNA group was much lower than the negative siRNA group,according to fluorescence microscopy results.The mRNA and protein levels of Bcl-XL in Bcl-XL siRNAstable transfectants were reduced to almost background level compared with negative siRNA transfectantsor untreated cells.(2)Changes in nucleus morphology was observed by AO staining nucleic and flowcytometry analysis,which showed that stable Bcl-XL siRNA transfectants have an increased spontaneousapoptosis (21.17%+1.26% vs.1.19%+0.18% and 1.56%+0.15% respectively,P<0.05 vs.negative siRNAor untreated control),siRNA targeting GFP or Bcl-XL genes can specifically suppress GFP or BcI-XLexpression in MGC-803 cells,and Bcl-XL siRNA can increase spontaneous apoptosis.Bcl-XL siRNA maybe a beneficial agent against human gastric adenocarcinoma.     

Green Fluorescent Protein (GFP) as a Reporter Gene for the Plant Pathogenic Oomycete Phytophthora ramorum

ABSTRACT. Transgenic Phytophthora ramorum strains that produce green fluorescent protein (GFP) constitutively were obtained after stable DNA integration using a polyethylene glycol and CaCl₂-based transformation protocol. Green fluorescent protein production was studied in developing colonies and in different propagules of the pathogen to evaluate its use in molecular and physiological studies. About 12% of the GFP transformants produced GFP to a level detectable by a confocal laser scanning microscope. Green fluorescent protein could be visualized in structures with vital protoplasm, such as hyphal tips and germinating cysts. In infection studies with Rhododendron , one of the GFP expressing strains showed aggressiveness equal to that of the corresponding non-labelled isolate. Thus, GFP could be used as a reporter gene in P. ramorum . Limitations of the technology are discussed.     

Analysis of osm-6, a gene that affects sensory cilium structure and sensory neuron function in Caenorhabditis elegans.

Mutation in the Caenorhabditis elegans gene osm-6 was previously shown to result in defects in the ultrastructure of sensory cilia and defects in chemosensory and mechanosensory behaviors. We have cloned osm-6 by transposon tagging and transformation rescue and have identified molecular lesions associated with five osm-6 mutations. The osm-6 gene encodes a protein that is 40% identical in amino acid sequence to a predicted mammalian protein of unknown function. We fused osm-6 with the gene for green fluorescent protein (GFP); the fusion gene rescued the osm-6 mutant phenotype and showed accumulation of GFP in ciliated sensory neurons exclusively. The OSM-6::GFP protein was localized to cytoplasm, including processes and dendritic endings where sensory cilia are situated. Mutations in other genes known to cause ciliary defects led to changes in the appearance of OSM-6::GFP in dendritic endings or, in the case of daf-19, reduced OSM-6::GFP accumulation. We conclude from an analysis of genetic mosaics that osm-6 acts cell autonomously in affecting cilium structure.     

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.     

A simple estimation of peroxisomal degradation with green fluorescent protein - an application for cell cycle analysis.

When nutrients are depleted from the environment, mammalian cells begin to degrade their own cytosol and organelles. This bulk protein degradation is mediated by autophagy. In this study, peroxisomes in living CHO-K1 cells were visualized by targeting the green fluorescent protein (GFP) tagged with a type 1 peroxisomal targeting signal. The nutrient-starved condition induced a decay of GFP fluorescence in the peroxisomes and autophagic inhibitors such as 3-methyladenine suppressed the decay of GFP fluorescence (13-60% of starvation). By double labeling the nuclear DNA and peroxisomal GFP, the autophagy specifically occurred at the G1 phase of the cell cycle and the autophagic inhibitors suppressed the G1 arrest. The vital stain technique with GFP is a very simple and useful marker to quantitatively estimate or to further study peroxisomal degradation.     

Expression of two heterologous proteins depends on the mode of expression: comparison of in vivo and in vitro methods

The yield of two proteins, avidin and green fluorescent protein (GFP), expressed from a modified Autographa californica nucleopolyhedrovirus (AcMNPV), was compared in Sf9 cell culture monolayer, Sf21 cell suspension culture and intact Spodoptera litura larvae. GFP expressed from the p10 promoter yielded up to 1.5% of total soluble protein in larvae, 20-fold higher than that in monolayer suspension culture. Avidin, expressed from the polh promoter, yielded up to 2.3% of total soluble protein in larvae, 10-fold higher than that in suspension culture and 40-fold higher than that in monolayers. Avidin expression did not affect amounts of GFP in dual-expressing baculovirus compared with those detected from a GFP-only expressing AcMNPV. A biotin-binding assay showed that all avidin expressed in larvae was fully active. Glycosylation patterns of chicken-avidin and Spodoptera-avidin were very similar, though the latter showed a proportion of partially glycosylated material.     

Endosperm-specific expression of green fluorescent protein driven by the hordein promoter is stably inherited in transgenic barley (Hordeum vulgare) plants

The expression of green fluorescent protein (GFP) and its inheritance were studied in transgenic barley (Hordeum vulgare L.) plants transformed with a synthetic green fluorescent protein gene [sgfp(S65T)] driven by either a rice actin promoter or a barley endosperm-specific d-hordein promoter. The gene encoding phosphinothricin acetyltransferase (bar), driven by the maize ubiquitin promoter and intron, was used as a selectable marker to identify transgenic tissues. Strong GFP expression driven by the rice actin promoter was observed in callus cells and in a variety of tissues of T0 plants transformed with the sgfp(S65T)-containing construct. GFP expression, driven by the rice actin promoter, was observed in 14 out of 17 independent regenerable transgenic callus lines; however, expression was gradually lost in T0 and later generation progeny of diploid lines. Stable GFP expression was observed in T2 progeny from only 6 out of the 14 (43%) independent GFP-expressing callus lines. Four of the 8 lines not expressing GFP in T2 progeny, lost GFP expression during T0 plant regeneration from calli; one lost GFP expression in the transition from the T0 to T1 generations and three lines were sterile. Similarly, expression of bar driven by the maize ubiquitin promoter was lost in T1 progeny; only 21 out of 26 (81%) independent lines were Basta-resistant. In contrast to actin-driven expression, GFP expression driven by the d-hordein promoter exhibited endosperm-specificity. All seven lines transformed with d-hordein-driven GFP (100%) expressed GFP in the T1 and T2 generations, regardless of ploidy levels, and expression segregated in a Mendelian fashion. We conclude that the sgfp(S65T) gene was successfully transformed into barley and that GFP expression driven by the d-hordein promoter was more stable in its inheritance pattern in T1 and T2 progeny than that driven by the rice actin promoter or the bar gene driven by the maize ubiquitin promoter.     

In vivo and in vitro protein solubility assays using split GFP

The rapid assessment of protein solubility is essential for evaluating expressed proteins and protein variants for use as reagents for downstream studies. Solubility screens based on antibody blots are complex and have limited screening capacity. Protein solubility screens using split beta-galactosidase in vivo and in vitro can perturb protein folding. Split GFP used for monitoring protein interactions folds poorly, and to overcome this limitation, we recently developed a protein-tagging system based on self-complementing split GFP derived from an exceptionally well folded variant of GFP termed 'superfolder GFP'. Here we present the step-by-step procedure of the solubility assay using split GFP. A 15-amino-acid GFP fragment, GFP 11, is fused to a test protein. The GFP 1-10 detector fragment is expressed separately. These fragments associate spontaneously to form fluorescent GFP. The fragments are soluble, and the GFP 11 tag has minimal effect on protein solubility and folding. We describe high-throughput protein solubility screens amenable both for in vivo and in vitro formats. The split-GFP system is composed of two vectors used in the same strain: pTET GFP 11 and pET GFP 1-10 (Fig. 1 and Supplementary Note online). The gene encoding the protein of interest is cloned into the pTET GFP 11 vector (resulting in an N-terminal fusion) and transformed into Escherichia coli BL21 (DE3) cells containing the pET GFP 1-10 plasmid. We also describe how this system can be used for selecting soluble proteins from a library of variants (Box 1). The large screening power of the in vivo assay combined with the high accuracy of the in vitro assay point to the efficiency of this two-step split-GFP tool for identifying soluble clones suitable for purification and downstream applications.