Recently, we have reported a new gfp gene isolated from Aequorea macrodactyla. The protein purified from expressed E. coli exhibited an excitation peak at 476 nm and an emission peak at 496 nm. However, the drawback of only maturing to fluorescence at low temperature limited its applications. In this paper, we further describe twelve mutants of GFPxm. Seven mutants produced enhanced fluorescence when expressed in E. coli at higher temperature (37 degrees C). After six hours of induction at 25 degrees C, 32 degrees C and 37 degrees C respectively, the relative fluorescent intensities of GFPxm16, GFPxm18 and GFPxm19 were higher than that of EGFP, moreover GFPxm16 and GFPxm163 could preserve high fluorescent intensity even expressed at 42 degrees C. Four mutants of the seven could reach high expression level in three kind of mammalian cells. Another 6 mutants had red-shift of excitation-emission maxima, and longest excitation-emission maxima were 514nm and 525nm. Another three mutants had two excitation peaks, and one mutant had only one UV-excitation peak. The most exciting result is the mutant of OFPxm with orange color. The mutant has an excitation peak at 509 nm and an emission peak at 523nm. 523nm is yellowish green but the protein is orange observed by eyes. The mutant could reach high expression level and matured at higher temperature but the fluorescent intensity was comparatively low because of low quantum yield.
Animals ; Electrophoresis, Polyacrylamide Gel ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Luminescent Proteins ; genetics ; metabolism ; Mutation ; Temperature

Fluorescence ; Host-Pathogen Interactions ; Luminescent Measurements ; methods ; Luminescent Proteins ; chemistry ; genetics ; metabolism ; Plant Diseases ; virology ; Plant Viruses ; chemistry ; genetics ; metabolism ; Plants ; chemistry ; genetics ; metabolism ; virology

The plasmodium of Physarum polycephalum is a suitable eukaryotic cell for cell cycle investigation, but there is no compatible transient expression system for the plasmodium. Using the promoter and terminator of ardC actin of Physarum polycephalum substituted the CMV IE and SV40 polyA of plasmid pDsRedl-N1, using cassette PardC-MCS-DsRed1-TardC substituted the cassette PardC-hph-TardC of plasmid pTB38, we constructed plasmids pXM1 and pXM2 for transient expression of red fluorescent protein (RFP) in Physarum polycephalum respectively. After reconstituting the transcription elongation factor homologous gene (pelf1) of Physarum polycephalum into the pXM2, we generated a plasmid pXM2-pelf1. After the plasmid pXM1, pXM2 and pXM2-pelf1 were electroporated into the plasmodium of Physarum polycephalum, we observed optimum RFP and PELF1-RFP expression under fluoroscope and confocal microscope between 24-48 h after electroporation, and found that ELF1-RFP expression was accumulated in nucleus of microplasmodium, the optimum electroporation parameters were 40 V/cm electric field, 1 ampere current, and 70 micros electric shock time. The results suggest that this expression system is qualified for transient expression of specific protein in plasmodium of Physarum polycephalum.
Actins ; genetics ; metabolism ; Electroporation ; Luminescent Proteins ; biosynthesis ; genetics ; Physarum polycephalum ; genetics ; metabolism ; Plasmids ; genetics ; metabolism ; Transcriptional Elongation Factors ; genetics

A stable dark variant separated from photobacterium phosphoreum (A2) was fixed in agar-gel membrane and immobilized onto an exposed end of a fiber-optic linked with bioluminometer. The variant could emit a luminescent signal in the presence of genotoxic agents, such as Mitomycin C (MC). The performance of this whole-cell optical fiber sensor system was examined as a function of several parameters, including gel probe thickness, bacterial cell density, and diameter of the fiber-optic core and working temperature. An optimal response to a model genotoxicant, Mitomycin C, was achieved with agar-bacterial gel membrane: the thickness of gel membrane was about 5 mm; the cell density of bacteria in gel membrane was about 2.0 x 10(7)/ml; the diameter of fiber-optic core was 5.0 mm; the working temperature was 25 degrees C. Under these optimized conditions, the response time was less than 10 h to Mitomycin C, with a lower detection threshold of 0.1 mg/L.
Biosensing Techniques ; Fiber Optic Technology ; Genetic Variation ; Luminescent Measurements ; Luminescent Proteins ; genetics ; Mitomycin ; pharmacology ; toxicity ; Optical Fibers ; Photobacterium ; genetics ; Transcription, Genetic ; drug effects

OBJECTIVETo study the expression and purification of a fusion protein of ricin A chain (RTA) and green fluorescent protein (GFP).

METHODSThe DNA sequence encoding ricin A chain was inserted into pEGFPC1 first to make the template sequence of the fusion protein. The fusion gene was amplified from the plasmid pEGFP-RTA by PCR, and directly subcloned into T vector. The fusion gene then was cloned into expression vector pET-28a(+), and the sequence was confirmed by sequencing. Expression was induced by IPTG in E. coli BL21(DE3). The fusion protein was purified by metal chelated affinity chromatography. The cytotoxicity of fusion protein was analyzed by the MTT assay in HepG2 and Hela cells.

RESULTSThe fusion protein of ricin A chain and GFP could be produced in E. coli transformed with the expression plasmid of pET-28a(+)-GFP-RTA. The molecular weight of the recombinant protein was measured by SDS-PAGE. The fusion protein showed a green fluorescence and had a similar cytotoxicity of RTA.

CONCLUSIONA recombinant fusion protein of RTA and GFP expressed in E. coli is possessed of similar biological activity of individual GFP and RTA, which could be used in study of the intracellular trafficking and translocation of RTA.

Escherichia coli ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; HeLa Cells ; Humans ; Luminescent Proteins ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Ricin ; genetics

This study was aimed to construct, package and purify the recombinant lentivirus vector carrying the firefly luciferase gene (FLUC) and red fluorescent protein gene (RFP) and to transfect the recombinant lentivirus into HeLa cells, so as to observe the expression levels of these two genes. The FLUC and RFP genes were amplified by RT-PCR and inserted in the lentiviral expression vector (pLenti-Bi-cistronic) to construct the lentiviral vector pLenti-FLUC-RFP. The viral particles were generated by cotransfection of 293T cells with pLenti-FLUC-RFP and three packaging vectors, and the virus titer was determined by calculating the percentage of RFP positive cells. After transfection of pLenti-FLUC-RFP into HeLa cells, the expression of RFP was observed by fluorescent microscopy, and the activity of FLUC was determined by luciferase reporter gene assay kit. The results showed that the inserting orientation of the RFP and FLUC genes in the lentiviral vector pLenti-FLUC-RFP were verified by restriction analysis. Targeted RFP and FLUC sequences were confirmed by DNA sequencing. The final titer obtained was 1×10(7)TU/ml. The expressions of RFP and FLUC were observed in the transfected HeLa cells. It is concluded that the pLenti-III-FLUC-RFP recombinant lentivirus vector carrying RFP gene and FLUC gene with high viral titer is constructed and packaged successfully, and provides experimental basis for studying dynamic distribution of mesenchymal stem cells in vivo.
Gene Expression ; Genes, Reporter ; Genetic Vectors ; HeLa Cells ; Humans ; Lentivirus ; genetics ; Luciferases, Firefly ; genetics ; Luminescent Proteins ; genetics ; Transfection

This study was purposed to construct a lentiviral vector encoding red fluorescent protein (DsRed) and transfect DsRed into EL4 cells for establishing mouse leukemia/lymphoma model expressing DsRed. The bicistronic SIN lentiviral transfer plasmid containing the genes encoding neo and internal ribosomal entry site-red fluorescent protein (IRES-DsRed) was constructed. Human embryonic kidney 293FT cells were co-transfected with the three plasmids by liposome method. The viral particles were collected and used to transfect EL4 cells, then the cells were selected by G418. The results showed that the plasmid pXZ208-neo-IRES-DsRed was constructed successfully, and the viral titer reached to 10(6) U/ml. EL4 cells were transfected by the viral solution efficiently. The transfected EL4 cells expressing DsRed survived in the final concentration 600 microg/ml of G418. The expression of DsRed in the transfected EL4 cells was demonstrated by fluorescence microscopy and flow cytometry. In conclusion, the EL4/DsRed cell line was established successfully.
Animals ; Cell Line, Tumor ; Flow Cytometry ; Genetic Vectors ; Lentivirus ; genetics ; Luminescent Proteins ; genetics ; Lymphoma ; genetics ; Mice ; Mice, Inbred C57BL ; Transfection

Most studies related to determining the expression profile of genes and specific promoters used histochemical localization of the reporter gene, gusA. While the histochemical method for visualizing gusA expression suffers from several limitations in the determination of gene expression and location, especially in the tissues with high background acitivty. To solve this problem, a transient expession vector pBI221-RFP/GFP, was constructed using GFP and RFP as double fluorescent marker genes. This vector used CaMV 35S promoter to drive GFP and determine the transforming efficiency. It analyzed expression profile of the target gene and promoter through the RFP activities of the tranformed tissues. Through the specific promoter AGPL1 from watermelon and E8 promoter from tomato, it is resistible to use this vector to study the expression patterns of promoters. Results indicated that the pBI221-RFP/GFP is a very efficient transient expression vector that can be verify the functions of the genes and promoters.
Citrullus ; genetics ; Gene Expression Regulation, Plant ; Genes, Plant ; Genes, Reporter ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Luminescent Agents ; metabolism ; Luminescent Proteins ; genetics ; metabolism ; Lycopersicon esculentum ; genetics ; Promoter Regions, Genetic

Gene Expression ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; Hepatocyte Growth Factor ; genetics ; Hepatocytes ; cytology ; metabolism ; Humans ; Luminescent Proteins ; genetics ; metabolism ; Microscopy, Fluorescence ; Recombinant Fusion Proteins ; genetics ; metabolism

OBJECTIVETo examine the potential function of NMDA receptor NR2A subunit C-terminus in assembling and surface expression of the receptor in HEK293 cells.

METHODSFive vectors GFP- NR2ADeltaC1- DeltaC5 were constructed for expressing N-terminally GFP-tagged NR2A with C-terminal deletion at different regions by using conventional techniques of molecular cloning. The deleted region for NR2ADeltaC1-Delta C5 was 897L-1017S, 1024D-1142P, 1149D-1347G, 1354S-1464V, and 897L-1464V. These plasmids were transfected alone or co-transfected with NR1-1a into HEK293 cells. The surface NMDA receptors were immuno-stained using rabbit antibody against GFP and Cy3 conjugated secondary antibody in living cells.

RESULTThe vectors GFP-NR2ADeltaC1-DeltaC5 were generated and all of them expressed GFP fluorescence in the transfected cells. Surface NMDA receptors were detected by immuno-labeling with anti-GFP in the cells co-transfected by NR1-1a and any one of GFP-NR2ADeltaC1-DeltaC5. However, no surface expression of NR2A proteins was found in the transfected cells with any one of these plasmids alone.

CONCLUSIONWithin the region downstream from the 897L of NR2A subunit, neither a particular domain directly interacted with ER retention domain in NR1-1a C1 cassette, nor that determining ER retention of NR2A subunit itself has been found, indicating that more complicated mechanisms might exist in which the subunit assembling and targeting to plasma membrane of NMDA receptors undergo.

Cell Line ; Gene Deletion ; Green Fluorescent Proteins ; Humans ; Luminescent Proteins ; metabolism ; Mutation ; Receptors, N-Methyl-D-Aspartate ; analysis ; genetics