Transfection of Cells with DNA Encoding a Visible Fluorescent Protein-Tagged Lipid-Binding Domain

2010 ◽  
Vol 2010 (7) ◽  
pp. pdb.prot5457-pdb.prot5457
Author(s):  
C. Schultz ◽  
A. B. Neef ◽  
T. W. Gadella ◽  
J. Goedhart
Keyword(s):  
Fluorescent Protein ◽  
Lipid Binding ◽  
Binding Domain ◽  
Dna Encoding

scholarly journals Specificity of the lipid-binding domain of apoC-II for the substrates and products of lipolysis

2001 ◽  
Vol 42 (4) ◽  
pp. 553-562
Author(s):  
M. Dahim ◽  
W.E. Momsen ◽  
M.M. Momsen ◽  
H.L. Brockman
Keyword(s):  
Lipid Binding ◽  
Binding Domain

scholarly journals Adaptation of the Endogenous Salmonella enterica Serovar Typhi clyA-Encoded Hemolysin for Antigen Export Enhances the Immunogenicity of Anthrax Protective Antigen Domain 4 Expressed by the Attenuated Live-Vector Vaccine Strain CVD 908-htrA

2004 ◽  
Vol 72 (12) ◽  
pp. 7096-7106 ◽  
Author(s):  
James E. Galen ◽  
Licheng Zhao ◽  
Magaly Chinchilla ◽  
Jin Yuan Wang ◽  
Marcela F. Pasetti ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Protective Antigen ◽  
Expression System ◽  
Dna Encoding ◽  
Protein Fusion ◽  
Vector Vaccine ◽  
Interleukin 5 ◽  
Export System ◽  
Domain 4

ABSTRACT Bacterial live-vector vaccines aim to deliver foreign antigens to the immune system and induce protective immune responses, and surface-expressed or secreted antigens are generally more immunogenic than cytoplasmic constructs. We hypothesize that an optimum expression system will use an endogenous export system to avoid the need for large amounts of heterologous DNA encoding additional proteins. Here we describe the cryptic chromosomally encoded 34-kDa cytolysin A hemolysin of Salmonella enterica serovar Typhi (ClyA) as a novel export system for the expression of heterologous antigens in the supernatant of attenuated Salmonella serovar Typhi live-vector vaccine strains. We constructed a genetic fusion of ClyA to the reporter green fluorescent protein and showed that in Salmonella serovar Typhi CVD 908-htrA, the fusion protein retains biological activity in both domains and is exported into the supernatant of an exponentially growing live vector in the absence of detectable bacterial lysis. The utility of ClyA for enhancing the immunogenicity of an otherwise problematic antigen was demonstrated by engineering ClyA fused to the domain 4 (D4) moiety of Bacillus anthracis protective antigen (PA). A total of 11 of 15 mice immunized intranasally with Salmonella serovar Typhi exporting the protein fusion manifested fourfold or greater rises in serum anti-PA immunoglobulin G, compared with only 1 of 16 mice immunized with the live vector expressing cytoplasmic D4 (P = 0.0002). In addition, the induction of PA-specific gamma interferon and interleukin 5 responses was observed in splenocytes. This technology offers exceptional versatility for enhancing the immunogenicity of bacterial live-vector vaccines.

scholarly journals Ca2+/calmodulin transfers the membrane-proximal lipid-binding domain of the v-SNARE synaptobrevin from cis to trans bilayers

2004 ◽  
Vol 101 (6) ◽  
pp. 1578-1583 ◽  
Author(s):  
L. de Haro ◽  
G. Ferracci ◽  
S. Opi ◽  
C. Iborra ◽  
S. Quetglas ◽  
...  
Keyword(s):  
Lipid Binding ◽  
Binding Domain

Interaction of azadirachtin with the lipid-binding domain: Suppression of lipid transportation in the silkworm, Bombyx mori

2018 ◽  
Vol 152 ◽  
pp. 62-68 ◽  
Author(s):  
Pratheep Thangaraj ◽  
Ramesh kumar Neelamegam ◽  
Kayalvizhi Nagarajan ◽  
Krishnan Muthukalingan
Keyword(s):  
Bombyx Mori ◽  
Lipid Binding ◽  
Binding Domain ◽  
Silkworm Bombyx Mori

scholarly journals Functional domains of interferon regulatory factor I (IRF-1)

1998 ◽  
Vol 335 (1) ◽  
pp. 147-157 ◽  
Author(s):  
Fred SCHAPER ◽  
Sabine KIRCHHOFF ◽  
Guido POSERN ◽  
Mario KÖSTER ◽  
André OUMARD ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Consensus Sequence ◽  
Binding Domain ◽  
Functional Domains ◽  
Dna Binding Domain ◽  
Transcriptional Activation Domain

Interferon (IFN) regulatory factors (IRFs) are a family of transcription factors among which are IRF-1, IRF-2, and IFN consensus sequence binding protein (ICSBP). These factors share sequence homology in the N-terminal DNA-binding domain. IRF-1 and IRF-2 are further related and have additional homologous sequences within their C-termini. Whereas IRF-2 and ICSBP are identified as transcriptional repressors, IRF-1 is an activator. In the present work, the identification of functional domains in murine IRF-1 with regard to DNA-binding, nuclear translocation, heterodimerization with ICSBP and transcriptional activation are demonstrated. The minimal DNA-binding domain requires the N-terminal 124 amino acids plus an arbitrary C-terminal extension. By using mutants of IRF-1 fusion proteins with green fluorescent protein and monitoring their distribution in living cells, a nuclear location signal (NLS) was identified and found to be sufficient for nuclear translocation. Heterodimerization was confirmed by a two-hybrid system adapted to mammalian cells. The heterodimerization domain in IRF-1 was defined by studies in vitroand was shown to be homologous with a sequence in IRF-2, suggesting that IRF-2 also heterodimerizes with ICSBP through this sequence. An acidic domain in IRF-1 was found to be required and to be sufficient for transactivation. Epitope mapping of IRF-1 showed that regions within the NLS, the heterodimerization domain and the transcriptional activation domain are exposed for possible contacts with interacting proteins.

Fluorescent protein-based reporters of the actin cytoskeleton in living plant cells: Fluorophore variant, actin binding domain, and promoter considerations

Cytoskeleton ◽  
2014 ◽  
Vol 71 (5) ◽  
pp. 311-327 ◽  
Author(s):  
Julia Dyachok ◽  
J. Alan Sparks ◽  
Fuqi Liao ◽  
Yuh-Shuh Wang ◽  
Elison B. Blancaflor
Keyword(s):  
Actin Cytoskeleton ◽  
Fluorescent Protein ◽  
Plant Cells ◽  
Binding Domain ◽  
Actin Binding ◽  
Living Plant ◽  
Actin Binding Domain

scholarly journals A lipid-binding domain in Wnt: a case of mistaken identity?

1999 ◽  
Vol 9 (19) ◽  
pp. R717-R719 ◽  
Author(s):  
Michael R. Barnes ◽  
Robert B. Russell ◽  
Richard R. Copley ◽  
Chris P. Ponting ◽  
Peer Bork ◽  
...  
Keyword(s):  
Lipid Binding ◽  
Binding Domain ◽  
Mistaken Identity

scholarly journals Identification of an 11-residue portion of CTP–phosphocholine cytidylyltransferase that is required for enzyme–membrane interactions

1997 ◽  
Vol 325 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Jilin YANG ◽  
Jinxia WANG ◽  
Irene TSEU ◽  
Maciej KULISZEWSKI ◽  
Wensu LEE ◽  
...  
Keyword(s):  
Oleic Acid ◽  
Fusion Proteins ◽  
Membrane Binding ◽  
Lipid Vesicles ◽  
Lipid Binding ◽  
Binding Domain ◽  
Membrane Interactions ◽  
Phosphocholine Cytidylyltransferase ◽  
N Terminus ◽  
L2 Cells

CTP–phosphocholine cytidylyltransferase (CT) is a key regulatory enzyme in the biosynthesis of phosphatidylcholine (PC) in many cells. Enzyme–membrane interactions appear to play an important role in CT activation. A putative membrane-binding domain appears to be located between residues 236 and 293 from the N-terminus. To map the membrane-binding domain more precisely, glutathione S-transferase fusion proteins were prepared that contained deletions of various domains in this putative lipid-binding region. The fusion proteins were assessed for their binding of [3H]PC/oleic acid vesicles. Fusion proteins encompassing residues 267–277 bound to PC/oleic acid vesicles, whereas fragments lacking this region exhibited no specific binding to the lipid vesicles. The membrane-binding characteristics of the CT fusion proteins were also examined using intact lung microsomes. Only fragments encompassing residues 267–277 competed with full-length 125I-labelled CT, expressed in recombinant Sf9 insect cells, for microsomal membrane binding. To investigate the role of this region in PC biosynthesis, A549 and L2 cells were transfected with cDNA for CT mutants under the control of a glucocorticoid-inducible long terminal repeat (LTR) promoter. Induction of CT mutants containing residues 267–277 in transfectants resulted in reduced PC synthesis. The decrease in PC synthesis was accompanied by a shift in endogenous CT activity from the particulate to the soluble fraction. Expression of CT mutants lacking this region in A549 and L2 cells did not affect PC formation and subcellular distribution of CT activity. These results suggest that the CT region located between residues 267 and 277 from the N-terminus is required for the interaction of CT with membranes.

Production of transgenic rabbit embryos through intracytoplasmic sperm injection

Zygote ◽  
2010 ◽  
Vol 18 (4) ◽  
pp. 301-307 ◽  
Author(s):  
Qiuyan Li ◽  
Jian Hou ◽  
Sheng Wang ◽  
Yongfu Chen ◽  
Xiao-Rong An
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Fluorescent Protein ◽  
Dna Uptake ◽  
Dna Encoding ◽  
Cell Stage ◽  
Transgenic Rabbit ◽  
Dna Attachment ◽  
Transgenic Embryos ◽  
Green Fluorescent ◽  
Rabbit Embryos

SummaryThe objective of this study was to test if intracytoplasmic sperm injection (ICSI)-mediated gene transfer was an effective method in the production of transgenic rabbit embryos. Rabbit sperm diluted in different media with various pH were treated by freezing without cryoprotectant, and their ability for DNA uptake was determined. In these experiments using production of transgenic rabbit embryos by ICSI, exogenous genes at three concentrations and of two conformation types were used. The rate of DNA association to the sperm seen by rhodamine-tagged DNA encoding green fluorescent protein (GFP) was 90.0%, 92.7%, 91.0%, 91.7%, and 92.3%, respectively in TCM199, DM, DPBS, CZB, and HCZB media. The DNA attachment to sperm was not affected by media pH within the range of 5.4–9.4 (p > 0.05). Expression of GFP first occurred at the 2-cell stage and continued to blastocyst formation. DNA concentration (between 5, 10, and 20 ng/μl) or conformation (linear and circular) had no effect on the production rate of transgenic embryos. These results indicated that genetically modified rabbit blastocysts can be efficiently produced by ICSI technique.

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