Application of Nanosecond Pulsed Electric Fields into HeLa Cells Expressing Enhanced Green Fluorescent Protein and Fluorescence Lifetime Microscopy

2012 ◽  
Vol 116 (36) ◽  
pp. 11159-11165 ◽  
Author(s):  
Kamlesh Awasthi ◽  
Takakazu Nakabayashi ◽  
Nobuhiro Ohta
Keyword(s):  
Green Fluorescent Protein ◽  
Electric Fields ◽  
Fluorescence Lifetime ◽  
Hela Cells ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Pulsed Electric Fields ◽  
Nanosecond Pulsed Electric Fields ◽  
Green Fluorescent

scholarly journals Deciphering the Role of Positions 145 and 165 in Fluorescence Lifetime Shortening in the EGFP Variants

Biomolecules ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1547
Author(s):  
Anastasia V. Mamontova ◽  
Aleksander M. Shakhov ◽  
Konstantin A. Lukyanov ◽  
Alexey M. Bogdanov
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescence Lifetime ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Important Determinant ◽  
Physicochemical Characteristics ◽  
High Brightness ◽  
Short Lifetime ◽  
Green Fluorescent

The bright ultimately short lifetime enhanced emitter (BrUSLEE) green fluorescent protein, which differs from the enhanced green fluorescent protein (EGFP) in three mutations, exhibits an extremely short fluorescence lifetime at a relatively high brightness. An important contribution to shortening the BrUSLEE fluorescence lifetime compared to EGFP is provided by the T65G substitution of chromophore-forming residue and the Y145M mutation touching the chromophore environment. Although the influence of the T65G mutation was studied previously, the role of the 145th position in determining the GFPs physicochemical characteristics remains unclear. In this work, we show that the Y145M substitution, both alone and in combination with the F165Y mutation, does not shorten the fluorescence lifetime of EGFP-derived mutants. Thus, the unlocking of Y145M as an important determinant of lifetime tuning is possible only cooperatively with mutations at position 65. We also show here that the introduction of a T65G substitution into EGFP causes complex photobehavior of the respective mutants in the lifetime domain, namely, the appearance of two fluorescent states with different lifetimes, preserved in any combination with the Y145M and F165Y substitutions.

scholarly journals Fluorescence lifetime images of green fluorescent protein in HeLa cells during TNF-α induced apoptosis

2009 ◽  
Vol 8 (6) ◽  
pp. 763 ◽  
Author(s):  
Toshiyuki Ito ◽  
Shugo Oshita ◽  
Takakazu Nakabayashi ◽  
Fan Sun ◽  
Masataka Kinjo ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescence Lifetime ◽  
Hela Cells ◽  
Fluorescent Protein ◽  
Tnf Α ◽  
Induced Apoptosis ◽  
Green Fluorescent

scholarly journals A functional chimaeric S-layer-enhanced green fluorescent protein to follow the uptake of S-layer-coated liposomes into eukaryotic cells

2004 ◽  
Vol 379 (2) ◽  
pp. 441-448 ◽  
Author(s):  
Nicola ILK ◽  
Seta KÜPCÜ ◽  
Gerald MONCAYO ◽  
Sigrid KLIMT ◽  
Rupert C. ECKER ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Hela Cells ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Laser Scanning Microscopy ◽  
Host Cells ◽  
Confocal Laser ◽  
Green Fluorescent ◽  
Egfp Fluorescence

The chimaeric gene encoding a C-terminally truncated form of the S-layer protein SbpA of Bacillus sphaericus CCM 2177 and the EGFP (enhanced green fluorescent protein) was ligated into plasmid pET28a and cloned and expressed in Escherichia coli. Just 1 h after induction of expression an intense EGFP fluorescence was detected in the cytoplasm of the host cells. Expression at 28 °C instead of 37 °C resulted in clearly increased fluorescence intensity, indicating that the folding process of the EGFP moiety was temperature sensitive. To maintain the EGFP fluorescence, isolation of the fusion protein from the host cells had to be performed in the presence of reducing agents. SDS/PAGE analysis, immunoblotting and N-terminal sequencing of the isolated and purified fusion protein confirmed the presence of both the S-layer protein and the EGFP moiety. The fusion protein had maintained the ability to self-assemble in suspension and to recrystallize on peptidoglycan-containing sacculi or on positively charged liposomes, as well as to fluoresce. Comparison of fluorescence excitation and emission spectra of recombinant EGFP and rSbpA31-1068/EGFP revealed identical maxima at 488 and 507 nm respectively. The uptake of liposomes coated with a fluorescent monomolecular protein lattice of rSbpA31-1068/EGFP into HeLa cells was studied by confocal laser-scanning microscopy. The major part of the liposomes was internalized within 2 h of incubation and entered the HeLa cells by endocytosis.

scholarly journals Enhanced Green Fluorescent Protein (GFP) fluorescence after polyelectrolyte caging

2006 ◽  
Vol 14 (21) ◽  
pp. 9815 ◽  
Author(s):  
Alberto Diaspro ◽  
Silke Krol ◽  
Barbara Campanini ◽  
Fabio Cannone ◽  
Giuseppe Chirico
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Gfp Fluorescence ◽  
Green Fluorescent

scholarly journals Development and Characterization of a cDNA-Launch Recombinant Simian Hemorrhagic Fever Virus Expressing Enhanced Green Fluorescent Protein: ORF 2b’ Is Not Required for In Vitro Virus Replication

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 632
Author(s):  
Yingyun Cai ◽  
Shuiqing Yu ◽  
Ying Fang ◽  
Laura Bollinger ◽  
Yanhua Li ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Lines ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Infectious Clone ◽  
Hemorrhagic Fever ◽  
Simian Hemorrhagic Fever Virus ◽  
Hemorrhagic Fever Virus ◽  
Green Fluorescent

Simian hemorrhagic fever virus (SHFV) causes acute, lethal disease in macaques. We developed a single-plasmid cDNA-launch infectious clone of SHFV (rSHFV) and modified the clone to rescue an enhanced green fluorescent protein-expressing rSHFV-eGFP that can be used for rapid and quantitative detection of infection. SHFV has a narrow cell tropism in vitro, with only the grivet MA-104 cell line and a few other grivet cell lines being susceptible to virion entry and permissive to infection. Using rSHFV-eGFP, we demonstrate that one cricetid rodent cell line and three ape cell lines also fully support SHFV replication, whereas 55 human cell lines, 11 bat cell lines, and three rodent cells do not. Interestingly, some human and other mammalian cell lines apparently resistant to SHFV infection are permissive after transfection with the rSHFV-eGFP cDNA-launch plasmid. To further demonstrate the investigative potential of the infectious clone system, we introduced stop codons into eight viral open reading frames (ORFs). This approach suggested that at least one ORF, ORF 2b’, is dispensable for SHFV in vitro replication. Our proof-of-principle experiments indicated that rSHFV-eGFP is a useful tool for illuminating the understudied molecular biology of SHFV.

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