Redesigning the type II' β -turn in green fluorescent protein to type I': Implications for folding kinetics and stability

2014 ◽  
Vol 82 (10) ◽  
pp. 2812-2822 ◽  
Author(s):  
Bharat Madan ◽  
Sriram Sokalingam ◽  
Govindan Raghunathan ◽  
Sun-Gu Lee
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Type I ◽  
Type Ii ◽  
Folding Kinetics ◽  
Green Fluorescent

scholarly journals Preparation of North American Type II PRRSV Infectious Clone Expressing Green Fluorescent Protein

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Liyue Wang ◽  
Kao Zhang ◽  
Hongyu Lin ◽  
Wenyan Li ◽  
Jiexia Wen ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
North American ◽  
Fluorescent Protein ◽  
Infectious Clone ◽  
Type Ii ◽  
Nsp2 Gene ◽  
Gfp Gene ◽  
Overlap Pcr ◽  
Green Fluorescent ◽  
North American Type

Porcine reproductive and respiratory syndrome virus (PRRSV) is still one of the most important infectious diseases threatening the swine industry. To construct North American type II PRRSV infectious clone containing green fluorescent protein (GFP) gene, we amplify gfp gene, flanked by PRRSV Nsp2 gene fragments upstream and downstream, using overlap PCR method from pcDNA-EF1-GFP plasmid and FL12 plasmid containing PRRSV infectious genome as the templates. The Nsp2 fragment-flanked gfp gene was inserted into Nsp2 gene of the FL12 plasmid bySpeI andXhoI sites to generate PRRSV infectious recombinant plasmid (FL12-GFP) containing gfp gene. The recombinant PRRSV expressing GFP (PRRSV-GFP) was rescued in baby hamster kidney-21 (BHK-21) cells by transfecting PRRSV mRNA synthesizedin vitroand amplified in Marc-145 cells. The PRRSV-GFP infectivity and replication capacity were identified. Results showed that, by adopting overlap PCR strategy, the gfp gene was successfully inserted into and fused with PRRSV Nsp2 gene in the PRRSV infectious clone plasmid FL-12 to generate FL12-GFP plasmid. The recombinant PRRSV-GFP was generated through transfecting PRRSV mRNA in BHK-2 cells. Like its parental virus, the recombinant PRRSV-GFP maintains its infectivity to Marc-145 cells and porcine alveolar macrophages (PAMs). This study provides essential conditions for further investigation on PRRSV.

A Caenorhabditis elegans type I TGF beta receptor can function in the absence of type II kinase to promote larval development

Development ◽  
2000 ◽  
Vol 127 (15) ◽  
pp. 3337-3347 ◽  
Author(s):  
C.V. Gunther ◽  
L.L. Georgi ◽  
D.L. Riddle
Keyword(s):  
Caenorhabditis Elegans ◽  
Fluorescent Protein ◽  
Type I ◽  
Receptor Complex ◽  
Type Ii ◽  
Precise Control ◽  
Dauer Larva ◽  
Protein Receptor ◽  
Morphogenetic Protein ◽  
Green Fluorescent

The daf-4 gene encodes a type II bone morphogenetic protein receptor in Caenorhabditis elegans that regulates dauer larva formation, body size and male tail patterning. The putative type I receptor partner for DAF-4 in regulating dauer larva formation is DAF-1. Genetic tests of the mechanism of activation of these receptors show that DAF-1 can signal in the absence of DAF-4 kinase activity. A daf-1 mutation enhances dauer formation in a daf-4 null background, whereas overexpression of daf-1 partially rescues a daf-4 mutant. DAF-1 alone cannot fully compensate for the loss of DAF-4 activity, indicating that nondauer development normally results from the activities of both receptors. DAF-1 signaling in the absence of a type II kinase is unique in the type I receptor family. The activity may be an evolutionary remnant, owing to daf-1's origin near the type I/type II divergence, or it may be an innovation that evolved in nematodes. daf-1 and daf-4 promoters both mediated expression of green fluorescent protein in the nervous system, indicating that a DAF-1/DAF-4 receptor complex may activate a neuronal signaling pathway. Signaling from a strong DAF-1/DAF-4 receptor complex or a weaker DAF-1 receptor alone may provide larvae with more precise control of the dauer/nondauer decision in a range of environmental conditions.

scholarly journals Generation of a Soluble Form of Human Endoglin Fused to Green Fluorescent Protein

2021 ◽  
Vol 22 (20) ◽  
pp. 11282
Author(s):  
Lidia Ruiz-Llorente ◽  
M. Cristina Vega ◽  
Francisco J. Fernández ◽  
Carmen Langa ◽  
Nicholas W. Morrell ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Extracellular Domain ◽  
Soluble Form ◽  
Migration And Invasion ◽  
Type I ◽  
Juxtamembrane Region ◽  
Green Fluorescent

Endoglin (Eng, CD105) is a type I membrane glycoprotein that functions in endothelial cells as an auxiliary receptor for transforming growth factor β (TGF-β)/bone morphogenetic protein (BMP) family members and as an integrin ligand, modulating the vascular pathophysiology. Besides the membrane-bound endoglin, there is a soluble form of endoglin (sEng) that can be generated by the action of the matrix metalloproteinase (MMP)-14 or -12 on the juxtamembrane region of its ectodomain. High levels of sEng have been reported in patients with preeclampsia, hypercholesterolemia, atherosclerosis and cancer. In addition, sEng is a marker of cardiovascular damage in patients with hypertension and diabetes, plays a pathogenic role in preeclampsia, and inhibits angiogenesis and tumor proliferation, migration, and invasion in cancer. However, the mechanisms of action of sEng have not yet been elucidated, and new tools and experimental approaches are necessary to advance in this field. To this end, we aimed to obtain a fluorescent form of sEng as a new tool for biological imaging. Thus, we cloned the extracellular domain of endoglin in the pEGFP-N1 plasmid to generate a fusion protein with green fluorescent protein (GFP), giving rise to pEGFP-N1/Eng.EC. The recombinant fusion protein was characterized by transient and stable transfections in CHO-K1 cells using fluorescence microscopy, SDS-PAGE, immunodetection, and ELISA techniques. Upon transfection with pEGFP-N1/Eng.EC, fluorescence was readily detected in cells, indicating that the GFP contained in the recombinant protein was properly folded into the cytosol. Furthermore, as evidenced by Western blot analysis, the secreted fusion protein yielded the expected molecular mass and displayed a specific fluorescent signal. The fusion protein was also able to bind to BMP9 and BMP10 in vitro. Therefore, the construct described here could be used as a tool for functional in vitro studies of the extracellular domain of endoglin.

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