scholarly journals mem-iLID, a fast and economic protein purification method

2021 ◽  
Author(s):  
Ruijing Tang ◽  
Shang Yang ◽  
Georg Nagel ◽  
Shiqiang Gao
Keyword(s):  
Protein Purification ◽  
Membrane Fraction ◽  
Biological Membrane ◽  
Xenopus Laevis Oocyte ◽  
Purification Method ◽  
Protein Dimerization ◽  
Protein Protein Interaction ◽  
Mild Conditions ◽  
Chromatography Columns ◽  
Harsh Conditions

Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based purifications, which require different chromatography columns and harsh conditions, such as acidic pH and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. Here we established an easy and fast purification method for soluble proteins under mild conditions, based on the light-induced protein dimerization system iLID, which regulates protein binding and release with light. We utilize the biological membrane, which can be easily separated by centrifugation, as the port to anchor the target proteins. In Xenopus laevis oocyte and Escherichia coli, the blue light-sensitive part of iLID, AsLOV2-SsrA, was targeted to the plasma membrane by different membrane anchors. The other part of iLID, SspB, was fused with the protein of interest (POI) and expressed in the cytosol. The SspB-POI can be captured to the membrane fraction through light-induced binding to AsLOV2-SsrA and then released purely to fresh buffer in the dark after simple centrifugation and washing. This method, named mem-iLID, is very flexible in scale and economic. We demonstrate the quickly obtained yield of two pure and fully functional enzymes: a DNA polymerase and a light-activated adenylyl cyclase. Furthermore, we also designed a new SspB mutant for better dissociation and less interference with the protein of interest, which could potentially facilitate other optogenetic manipulations of protein-protein interaction.

Pt-Pd Nanoalloy for the Unprecedented Activation of Carbon-Fluorine Bond at Low Temperature

2019 ◽  
Author(s):  
Raghu Nath Dhital ◽  
keigo nomura ◽  
Yoshinori Sato ◽  
Setsiri Haesuwannakij ◽  
Masahiro Ehara ◽  
...  
Keyword(s):  
Activation Energy ◽  
Low Temperature ◽  
Dft Calculations ◽  
Bond Activation ◽  
Mild Conditions ◽  
Selective Transformation ◽  
Rate Determining Step ◽  
Highly Active ◽  
Harsh Conditions ◽  
Reaction Profile

Carbon-Fluorine (C-F) bonds are considered the most inert organic functionality and their selective transformation under mild conditions remains challenging. Herein, we report a highly active Pt-Pd nanoalloy as a robust catalyst for the transformation of C-F bonds into C-H bonds at low temperature, a reaction that often required harsh conditions. The alloying of Pt with Pd is crucial to activate C-F bond. The reaction profile kinetics revealed that the major source of hydrogen in the defluorinated product is the alcoholic proton of 2-propanol, and the rate-determining step is the reduction of the metal upon transfer of the <i>beta</i>-H from 2-propanol. DFT calculations elucidated that the key step is the selective oxidative addition of the O-H bond of 2-propanol to a Pd center prior to C-F bond activation at a Pt site, which crucially reduces the activation energy of the C-F bond. Therefore, both Pt and Pd work independently but synergistically to promote the overall reaction

A simplified protein purification method through nickel cleavage of the recombinant protein from the Escherichia coli cell surface

The Analyst ◽  
2020 ◽  
Vol 145 (19) ◽  
pp. 6227-6231
Author(s):  
Shanqing Huang ◽  
Tianbiao Wei ◽  
Wanxing Sha ◽  
Qingyuan Hu ◽  
Yingying Zhang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Cell Surface ◽  
Protein Purification ◽  
Target Protein ◽  
Coli Cell ◽  
Purification Method

A remarkable simple protein purification method is developed through nickel cleaving the target protein off the Escherichia coli cell surface.

Pt-Pd Nanoalloy for the Unprecedented Activation of Carbon-Fluorine Bond at Low Temperature

2019 ◽  
Author(s):  
Raghu Nath Dhital ◽  
keigo nomura ◽  
Yoshinori Sato ◽  
Setsiri Haesuwannakij ◽  
Masahiro Ehara ◽  
...  
Keyword(s):  
Activation Energy ◽  
Low Temperature ◽  
Dft Calculations ◽  
Bond Activation ◽  
Mild Conditions ◽  
Selective Transformation ◽  
Rate Determining Step ◽  
Highly Active ◽  
Harsh Conditions ◽  
Reaction Profile

Carbon-Fluorine (C-F) bonds are considered the most inert organic functionality and their selective transformation under mild conditions remains challenging. Herein, we report a highly active Pt-Pd nanoalloy as a robust catalyst for the transformation of C-F bonds into C-H bonds at low temperature, a reaction that often required harsh conditions. The alloying of Pt with Pd is crucial to activate C-F bond. The reaction profile kinetics revealed that the major source of hydrogen in the defluorinated product is the alcoholic proton of 2-propanol, and the rate-determining step is the reduction of the metal upon transfer of the <i>beta</i>-H from 2-propanol. DFT calculations elucidated that the key step is the selective oxidative addition of the O-H bond of 2-propanol to a Pd center prior to C-F bond activation at a Pt site, which crucially reduces the activation energy of the C-F bond. Therefore, both Pt and Pd work independently but synergistically to promote the overall reaction.

scholarly journals Homotypic interactions of chicken GATA-1 can mediate transcriptional activation.

1995 ◽  
Vol 15 (3) ◽  
pp. 1353-1363 ◽  
Author(s):  
H Y Yang ◽  
T Evans
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Regulatory Elements ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Protein Dimerization ◽  
Protein Protein Interaction ◽  
Functional Consequences ◽  
Homotypic Interactions

We used a one-hybrid system to replace precisely the finger II chicken GATA-1 DNA-binding domain with the binding domain of bacterial repressor protein LexA. The LexA DNA-binding domain lacks amino acids that function for transcriptional activation, nuclear localization, or protein dimerization. This allowed us to analyze activities of GATA-1 sequences distinct from DNA binding. We found that strong transcriptional activating sequences that function independently of finger II are present in GATA-1. Sequences including finger I contain an independent nuclear localizing function. Our data are consistent with cooperative binding of two LexA-GATA-1 hybrid proteins on a palindromic operator. The sensitivity of our transcription assay provides the first evidence that GATA-1 can make homotypic interactions in vivo. The ability of a non-DNA-binding form of GATA-1 to activate gene expression by targeting to a bound GATA-1 derivative further supports the notion that GATA-1-GATA-1 interactions may have functional consequences. A coimmunoprecipitation assay was used to demonstrate that GATA-1 multimeric complexes form in solution by protein-protein interaction. The novel ability of GATA-1 to interact homotypically may be important for the formation of higher-order structures among distant regulatory elements that share binding sites for this transcription factor. We also used the system to test the ability of GATA-1 to interact heterotypically with other activators.

scholarly journals 18F-Labeling Using Click Cycloadditions

2014 ◽  
Vol 2014 ◽  
pp. 1-16 ◽  
Author(s):  
Kathrin Kettenbach ◽  
Hanno Schieferstein ◽  
Tobias L. Ross
Keyword(s):  
Emission Tomography ◽  
Complex Structures ◽  
Direct Introduction ◽  
New Techniques ◽  
Click Reactions ◽  
Mild Conditions ◽  
Positron Emission ◽  
Recent Trends ◽  
Prosthetic Groups ◽  
Harsh Conditions

Due to expanding applications of positron emission tomography (PET) there is a demand for developing new techniques to introduce fluorine-18 (t1/2=109.8 min). Considering that most novel PET tracers are sensitive biomolecules and that direct introduction of fluorine-18 often needs harsh conditions, the insertion of18F in those molecules poses an exceeding challenge. Two major challenges during18F-labeling are a regioselective introduction and a fast and high yielding way under mild conditions. Furthermore, attention has to be paid to functionalities, which are usually present in complex structures of the target molecule. The Cu-catalyzed azide-alkyne cycloaddition (CuAAC) and several copper-free click reactions represent such methods for radiolabeling of sensitive molecules under the above-mentioned criteria. This minireview will provide a quick overview about the development of novel18F-labeled prosthetic groups for click cycloadditions and will summarize recent trends in copper-catalyzed and copper-free click18F-cycloadditions.

scholarly journals Release of Cationic Drugs from Charcoal

Materials ◽  
2019 ◽  
Vol 12 (4) ◽  
pp. 683
Author(s):  
Chiara Di Ruocco ◽  
Maria Acocella ◽  
Gaetano Guerra
Keyword(s):  
Hydrogen Peroxide ◽  
Aqueous Solutions ◽  
Uv Spectra ◽  
Wide Angle ◽  
X Ray Diffraction ◽  
X Ray ◽  
Acidic Solutions ◽  
Cationic Drugs ◽  
Mild Conditions ◽  
Harsh Conditions

The goal of this research is to improve preparation of charcoal adducts in a manner suitable for cationic drug release, possibly using an eco-friendly procedure. Charcoal, widely commercialized for human ingestion, is oxidized by hydrogen peroxide in mild conditions. Adducts of a cationic drug (lidocaine hydrochloride, a medication used as local anesthetic) with charcoal are prepared after basification of charcoal and characterized mainly by elemental analysis, wide-angle X-ray diffraction, infrared spectroscopy and thermogravimetry. The drug in the prepared adducts is present in amount close to 30% by weight and can be readily released to both neutral and acidic aqueous solutions. Cation release, as studied by UV spectra of aqueous solutions, is faster in acidic solutions and is faster than for adducts with graphite oxide, which can be prepared only in harsh conditions.

scholarly journals Membrane Binding Promotes Annexin A2 Oligomerization

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1169 ◽  
Author(s):  
Anna Lívia Linard Matos ◽  
Sergej Kudruk ◽  
Johanna Moratz ◽  
Milena Heflik ◽  
David Grill ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Biological Membrane ◽  
Membrane Binding ◽  
Annexin A2 ◽  
Protein Protein Interactions ◽  
Mutant Proteins ◽  
Lipid Domain ◽  
Protein Protein Interaction ◽  
Membrane Bound ◽  
Charged Membranes

Annexin A2 (AnxA2) is a cytosolic Ca2+ regulated membrane binding protein that can induce lipid domain formation and plays a role in exocytosis and endocytosis. To better understand the mode of annexin-membrane interaction, we analyzed membrane-bound AnxA2 assemblies by employing a novel 3-armed chemical crosslinker and specific AnxA2 mutant proteins. Our data show that AnxA2 forms crosslinkable oligomers upon binding to membranes containing negatively charged phospholipids. AnxA2 mutants with amino acid substitutions in residues predicted to be involved in lateral protein–protein interaction show compromised oligomer formation, albeit still being capable of binding to negatively charged membranes in the presence of Ca2+. These results suggest that lateral protein–protein interactions are involved in the formation of AnxA2 clusters on a biological membrane.

scholarly journals Photochemical site-selective synthesis of [70]methanofullerenes

2016 ◽  
Vol 52 (86) ◽  
pp. 12733-12736 ◽  
Author(s):  
Sara Vidal ◽  
Marta Izquierdo ◽  
Wai Kit Law ◽  
Kui Jiang ◽  
Salvatore Filippone ◽  
...  
Keyword(s):  
Selective Synthesis ◽  
Mild Conditions ◽  
Site Selective ◽  
Harsh Conditions

Methanofullerenes such as the well-known [70]PCBM are commonly synthesized under harsh conditions to obtain the product as a mixture of site-isomers (namely α, β and minor γ) due to the D5h symmetry of the C70 cage. α-[70]siteisomers are selectively prepared under light and mild conditions with minor or negligible β-[70]siteisomers formation.

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