scholarly journals Bifunctional fusion proteins containing the sequence of the bradykinin homologue maximakinin: activities at the rat bradykinin B2 receptor

2018 ◽  
Vol 96 (5) ◽  
pp. 459-470 ◽  
Author(s):  
Xavier Charest-Morin ◽  
Robert Lodge ◽  
François Marceau
Keyword(s):  
Fusion Proteins ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Protein Denaturation ◽  
Epifluorescence Microscopy ◽  
Terminal Sequence ◽  
Protein Ligands ◽  
C Terminus ◽  
Bona Fide ◽  
Green Fluorescent

To support bradykinin (BK) B2 receptor (B2R) detection and therapeutic stimulation, we developed and characterized fusion proteins consisting of the BK homolog maximakinin (MK), or variants, positioned at the C-terminus of functional proteins (enhanced green fluorescent protein (EGFP), the peroxidase APEX2, or human serum albumin (HSA)). EGFP-MK loses its reactivity with anti-BK antibodies and molecular mass as it progresses in the endosomal tract of cells expressing rat B2Rs (immunoblots, epifluorescence microscopy). APEX2-(NG)15-MK is a bona fide agonist of the rat, but not of the human B2R (calcium and c-Fos signaling) and is compatible with the cytochemistry reagent TrueBlue (microscopy), a luminol-based reagent, or 3,3′,5,5′-tetramethylbenzidine (luminescence or colourimetric B2R detection, cell well plate format). APEX2-(NG)15-MK is a non-isotopic ligand suitable for drug discovery via binding competition. Affinity-purified secreted forms of HSA fused with peptides possessing the C-terminal MK or BK sequence failed to stimulate the rat B2R in the concentration range of 50–600 nmol/L. However, the non-secreted construction myc-HSA-MK is a B2R agonist, indicating that protein denaturation made the C-terminal sequence available for receptor binding. Fusion protein ligands of the B2R are stable but subjected to slow intracellular inactivation, strong species specificity, and possible steric hindrance between the receptor and large proteins.

scholarly journals The C Terminus of Peripherin/rds Participates in Rod Outer Segment Targeting and Alignment of Disk Incisures

2004 ◽  
Vol 15 (4) ◽  
pp. 2027-2037 ◽  
Author(s):  
Beatrice M. Tam ◽  
Orson L. Moritz ◽  
David S. Papermaster
Keyword(s):  
Fusion Proteins ◽  
Outer Segment ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Membrane Domains ◽  
Rod Outer Segment ◽  
Retinal Degenerations ◽  
C Terminus ◽  
Green Fluorescent

Protein targeting is essential for domain specialization in polarized cells. In photoreceptors, three distinct membrane domains exist in the outer segment: plasma membrane, disk lamella, and disk rim. Peripherin/retinal degeneration slow (rds) and rom-1 are photoreceptor-specific members of the transmembrane 4 superfamily of transmembrane proteins, which participate in disk morphogenesis and localize to rod outer segment (ROS) disk rims. We examined the role of their C termini in targeting by generating transgenic Xenopus laevis expressing green fluorescent protein (GFP) fusion proteins. A GFP fusion containing residues 317-336 of peripherin/rds localized uniformly to disk membranes. A longer fusion (residues 307-346) also localized to the ROS but exhibited higher affinity for disk rims than disk lamella. In contrast, the rom-1 C terminus did not promote ROS localization. The GFP-peripherin/rds fusion proteins did not immunoprecipitate with peripherin/rds or rom-1, suggesting this region does not form intermolecular interactions and is not involved in subunit assembly. Presence of GFP-peripherin/rds fusions correlated with disrupted incisures, disordered ROS tips, and membrane whorls. These abnormalities may reflect competition of the fusion proteins for other proteins that interact with peripherin/rds. This work describes novel roles for the C terminus of peripherin/rds in targeting and maintaining ROS structure and its potential involvement in inherited retinal degenerations.

scholarly journals Marker Fusion Tagging, a New Method for Production of Chromosomally Encoded Fusion Proteins

2010 ◽  
Vol 9 (5) ◽  
pp. 827-830 ◽  
Author(s):  
Julian Lai ◽  
Seng Kah Ng ◽  
Fang Fang Liu ◽  
Rajesh Narhari Patkar ◽  
Yanfen Lu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Fusion Proteins ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Spindle Pole ◽  
Hygromycin B ◽  
Content Type ◽  
Spindle Pole Bodies ◽  
New Gene ◽  
Green Fluorescent

ABSTRACT A new gene-tagging method (marker fusion tagging [MFT]) is demonstrated for Neurospora crassa and Magnaporthe oryzae. Translational fusions between the hygromycin B resistance gene and various markers are inserted into genes of interest by homologous recombination to produce chromosomally encoded fusion proteins. This method can produce tags at any position and create deletion alleles that maintain N- and C-terminal sequences. We show the utility of MFT by producing enhanced green fluorescent protein (EGFP) tags in proteins localized to nuclei, spindle pole bodies, septal pore plugs, Woronin bodies, developing septa, and the endoplasmic reticulum.

scholarly journals In Vivo Inactivation of the Mycobacterial Integral Membrane Stearoyl Coenzyme A Desaturase DesA3 by a C-Terminus-Specific Degradation Process

2008 ◽  
Vol 190 (20) ◽  
pp. 6686-6696 ◽  
Author(s):  
Yong Chang ◽  
Gary E. Wesenberg ◽  
Craig A. Bingman ◽  
Brian G. Fox
Keyword(s):  
Coenzyme A ◽  
Fluorescent Protein ◽  
Degradation Process ◽  
Side Chains ◽  
Terminal Sequence ◽  
C Terminus ◽  
A Cell ◽  
Essential Enzyme ◽  
Green Fluorescent

ABSTRACT DesA3 (Rv3229c) from Mycobacterium tuberculosis is a membrane-bound stearoyl coenzyme A Δ9 desaturase that reacts with the oxidoreductase Rv3230c to produce oleic acid. This work provides evidence for a mechanism used by mycobacteria to regulate this essential enzyme activity. DesA3 expressed as a fusion with either a C-terminal His6 or c-myc tag had consistently higher activity and stability than native DesA3 having the native C-terminal sequence of LAA, which apparently serves as a binding determinant for a mycobacterial protease/degradation system directed at DesA3. Fusion of only the last 12 residues of native DesA3 to the C terminus of green fluorescent protein (GFP) was sufficient to make GFP unstable. Furthermore, the comparable C-terminal sequence from the Mycobacterium smegmatis DesA3 homolog Msmeg_1886 also conferred instability to the GFP fusion. Systematic examination revealed that residues with charged side chains, large nonpolar side chains, or no side chain at the last two positions were most important for stabilizing the construct, while lesser effects were observed at the third-from-last position. Using these rules, a combinational substitution of the last three residues of DesA3 showed that either DKD or LEA gave the best enhancement of stability for the modified GFP in M. smegmatis. Moreover, upon mutagenesis of LAA at the C terminus in native DesA3 to either of these tripeptides, the modified enzyme had enhanced catalytic activity and stability. Since many proteases are conserved within bacterial families, it is reasonable that M. tuberculosis will use a similar C-terminal degradation system to posttranslationally regulate the activity of DesA3 and other proteins. Application of these rules to the M. tuberculosis genome revealed that ∼10% the proteins encoded by essential genes may be susceptible to C-terminal proteolysis. Among these, an annotation is known for less than half, underscoring a general lack of understanding of proteins that have only temporal existence in a cell.

scholarly journals Cryoelectron Microscopy of Protein IX-Modified Adenoviruses Suggests a New Position for the C Terminus of Protein IX

2006 ◽  
Vol 80 (23) ◽  
pp. 11881-11886 ◽  
Author(s):  
Michael P. Marsh ◽  
Samuel K. Campos ◽  
Matthew L. Baker ◽  
Christopher Y. Chen ◽  
Wah Chiu ◽  
...  
Keyword(s):  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Indirect Evidence ◽  
Human Adenovirus ◽  
Cryoelectron Microscopy ◽  
Delivery Vector ◽  
C Terminus ◽  
Clinical Gene Therapy ◽  
Green Fluorescent ◽  
Protein Ix

ABSTRACT Recombinant human adenovirus is a useful gene delivery vector for clinical gene therapy. Minor capsid protein IX of adenovirus has been of recent interest since multiple studies have shown that modifications can be made to its C terminus to alter viral tropism or add molecular tags and/or reporter proteins. We examined the structure of an engineered adenovirus displaying the enhanced green fluorescent protein (EGFP) fused to the C terminus of protein IX. Cryoelectron microscopy and reconstruction localized the C-terminal EGFP fusion between the H2 hexon and the H4 hexon, positioned between adjacent facets, directly above the density previously assigned as protein IIIa. The original assignment of IIIa was based largely on indirect evidence, and the data presented herein support the reassignment of the IIIa density as protein IX.

Factor VIII-eGFP fusion proteins with preserved functional activity for the analysis of the early secretory pathway of factor VIII

2009 ◽  
Vol 102 (11) ◽  
pp. 925-935 ◽  
Author(s):  
Stefan Heinz ◽  
Jörg Schüttrumpf ◽  
Jeremy Simpson ◽  
Rainer Pepperkok ◽  
Gerry Nicolaes ◽  
...  
Keyword(s):  
Factor Viii ◽  
Fusion Proteins ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Recombinant Expression ◽  
Early Secretory Pathway ◽  
C Terminus ◽  
Living Organisms ◽  
Green Fluorescent

SummaryConsidering the difficulty in detecting factor (F)VIII in vivo, fluorescently labelled FVIII protein provides a tool to analyse the intracellular localisation, bio distribution, and pharmacokinetics of the protein in living organisms. Here, we report the use of FVIII full length and B-domain deleted proteins, fused to enhanced green fluorescent protein (eGFP) at the C-terminus of the coagulation protein via a nine amino acid spanning linker. Comparison of the FVIII-eGFP fusion proteins to their unlabelled counterparts showed no impairment with respect to recombinant expression levels, intracellular processing, specific coagulant activity and decay at physiological temperature. Confocal live cell imaging demonstrated ER-Golgi-transport of B-domain deleted FVIII-eGFP in vesicular tubular carriers. Using temperature blocks and release experiments, imaging of FVIII-eGFP fusion proteins enabled for the first time the visualisation of the early secretory pathway of B-domain deleted FVIII in living cells and in particular highlighted the apparent deficit of active transport carriers, an observation consistent with the low rates of FVIII secretion seen in recombinant expression systems.

scholarly journals Functional Expression and Subcellular Localization of the Aflatoxin Pathway Enzyme Ver-1 Fused to Enhanced Green Fluorescent Protein

2008 ◽  
Vol 74 (20) ◽  
pp. 6385-6396 ◽  
Author(s):  
Sung-Yong Hong ◽  
John E. Linz
Keyword(s):  
Green Fluorescent Protein ◽  
Subcellular Localization ◽  
Fusion Proteins ◽  
Laser Scanning ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Substrate Surface ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Green Fluorescent

ABSTRACT Aflatoxin, a mycotoxin synthesized by Aspergillus spp., is among the most potent naturally occurring carcinogens known. Little is known about the subcellular organization of aflatoxin synthesis. Previously, we used transmission electron microscopy and immunogold labeling to demonstrate that the late aflatoxin enzyme OmtA localizes primarily to vacuoles in fungal cells on the substrate surface of colonies. In the present work, we monitored subcellular localization of Ver-1 in real time in living cells. Aspergillus parasiticus strain CS10-N2 was transformed with plasmid constructs that express enhanced green fluorescent protein (EGFP) fused to Ver-1. Analysis of transformants demonstrated that EGFP fused to Ver-1 at either the N or C terminus functionally complemented nonfunctional Ver-1 in recipient cells. Western blot analysis detected predominantly full-length Ver-1 fusion proteins in transformants. Confocal laser scanning microscopy demonstrated that Ver-1 fusion proteins localized in the cytoplasm and in the lumen of up to 80% of the vacuoles in hyphae grown for 48 h on solid media. Control EGFP (no Ver-1) expressed in transformants was observed in only 13% of the vacuoles at this time. These data support a model in which middle and late aflatoxin enzymes are synthesized in the cytoplasm and transported to vacuoles, where they participate in aflatoxin synthesis.

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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