scholarly journals Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

10.3791/57069 ◽  
2018 ◽  
Author(s):  
Robert Serfling ◽  
Lisa Seidel ◽  
Thore Böttke ◽  
Irene Coin
Keyword(s):  
Mammalian Cells ◽  
Chemical Probes ◽  
Bioorthogonal Chemistry ◽  
Genetic Incorporation

scholarly journals A Genetically Encoded Picolyl Azide for Improved Live Cell Copper Click Labeling

2021 ◽  
Vol 9 ◽  
Author(s):  
Birthe Meineke ◽  
Johannes Heimgärtner ◽  
Alexander J. Craig ◽  
Michael Landreh ◽  
Lindon W. K. Moodie ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Stop Codon ◽  
Bioorthogonal Chemistry ◽  
Bioorthogonal Reaction ◽  
Noncanonical Amino Acids ◽  
Selective Reactivity ◽  
Direct Incorporation ◽  
Chelating Groups ◽  
Amber Suppression

Bioorthogonal chemistry allows rapid and highly selective reactivity in biological environments. The copper-catalyzed azide–alkyne cycloaddition (CuAAC) is a classic bioorthogonal reaction routinely used to modify azides or alkynes that have been introduced into biomolecules. Amber suppression is an efficient method for incorporating such chemical handles into proteins on the ribosome, in which noncanonical amino acids (ncAAs) are site specifically introduced into the polypeptide in response to an amber (UAG) stop codon. A variety of ncAA structures containing azides or alkynes have been proven useful for performing CuAAC chemistry on proteins. To improve CuAAC efficiency, biologically incorporated alkyne groups can be reacted with azide substrates that contain copper-chelating groups. However, the direct incorporation of copper-chelating azides into proteins has not been explored. To remedy this, we prepared the ncAA paz-lysine (PazK), which contains a picolyl azide motif. We show that PazK is efficiently incorporated into proteins by amber suppression in mammalian cells. Furthermore, PazK-labeled proteins show improved reactivity with alkyne reagents in CuAAC.

Genetic Incorporation of Multiple Unnatural Amino Acids into Proteins in Mammalian Cells

2013 ◽  
Vol 125 (52) ◽  
pp. 14330-14333 ◽  
Author(s):  
Han Xiao ◽  
Abhishek Chatterjee ◽  
Sei-hyun Choi ◽  
Krishna M. Bajjuri ◽  
Subhash C. Sinha ◽  
...  
Keyword(s):  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Mammalian Cells ◽  
Genetic Incorporation

Genetic incorporation of unnatural amino acids into proteins in mammalian cells

2007 ◽  
Vol 4 (3) ◽  
pp. 239-244 ◽  
Author(s):  
Wenshe Liu ◽  
Ansgar Brock ◽  
Shuo Chen ◽  
Shuibing Chen ◽  
Peter G Schultz
Keyword(s):  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Mammalian Cells ◽  
Genetic Incorporation

Genetic Incorporation of Multiple Unnatural Amino Acids into Proteins in Mammalian Cells

2013 ◽  
Vol 52 (52) ◽  
pp. 14080-14083 ◽  
Author(s):  
Han Xiao ◽  
Abhishek Chatterjee ◽  
Sei-hyun Choi ◽  
Krishna M. Bajjuri ◽  
Subhash C. Sinha ◽  
...  
Keyword(s):  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Mammalian Cells ◽  
Genetic Incorporation

Bioorthogonal chemistry for selective recognition, separation and killing bacteria over mammalian cells

2016 ◽  
Vol 52 (17) ◽  
pp. 3482-3485 ◽  
Author(s):  
Zhenhua Li ◽  
Zhen Liu ◽  
Zhaowei Chen ◽  
Enguo Ju ◽  
Wei Li ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Mammalian Cells ◽  
Selective Recognition ◽  
Bioorthogonal Chemistry ◽  
New Strategy

We report a new strategy for selective recognition, separation and killing bacteria using metabolic engineering and bioorthogonal chemistry.

Genetic Incorporation of a Photo-Crosslinkable Amino Acid Reveals Novel Protein Complexes with GRB2 in Mammalian Cells

2011 ◽  
Vol 406 (2) ◽  
pp. 343-353 ◽  
Author(s):  
Nobumasa Hino ◽  
Masaaki Oyama ◽  
Aya Sato ◽  
Takahito Mukai ◽  
Fumie Iraha ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mammalian Cells ◽  
Protein Complexes ◽  
Genetic Incorporation ◽  
Novel Protein

scholarly journals Genetic encoding of a highly photostable, long lifetime fluorescent amino acid for imaging in mammalian cells

2021 ◽  
Author(s):  
Chloe M. Jones ◽  
D. Miklos Robkis ◽  
Robert J. Blizzard ◽  
Mika Munari ◽  
Yarra Venkatesh ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Mammalian Cells ◽  
Trna Synthetase ◽  
Fluorescence Lifetime Imaging ◽  
High Quantum Yield ◽  
Lifetime Imaging ◽  
Genetic Encoding ◽  
Long Lifetime ◽  
Fluorescent Amino Acid ◽  
Genetic Incorporation

Acridonylalanine (Acd) is photostable, with a high quantum yield and long fluorescence lifetime in water. An evolved tRNA synthetase (RS) enables genetic incorporation of Acd in mammalian cells and its use in fluorescence lifetime imaging microscopy.

Chemical Probes for the Rapid Detection of Fatty-Acylated Proteins in Mammalian Cells

2007 ◽  
Vol 129 (10) ◽  
pp. 2744-2745 ◽  
Author(s):  
Howard C. Hang ◽  
Ernst-Jan Geutjes ◽  
Gijsbert Grotenbreg ◽  
Annette M. Pollington ◽  
Marie Jose Bijlmakers ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Mammalian Cells ◽  
Chemical Probes ◽  
Acylated Proteins

Ultrastructural Alterations in Hela Cells Induced by Spider Venom

1967 ◽  
Vol 25 ◽  
pp. 20-21
Author(s):  
Dale E. McClendon ◽  
Paul N. Morgan ◽  
Bernard L. Soloff
Keyword(s):  
Growth Medium ◽  
Mammalian Cells ◽  
Carcinoma Cells ◽  
Venom Protein ◽  
Sterile Saline ◽  
Carbon Coated ◽  
Available Information ◽  
Loxosceles Reclusa ◽  
Essential Growth ◽  
Solution Cultures

It has been observed that minute amounts of venom from the brown recluse spider, Loxosceles reclusa, are capable of producing cytotoxic changes in cultures of certain mammalian cells (Morgan and Felton, 1965). Since there is little available information concerning the effect of venoms on susceptible cells, we have attempted to characterize, at the electron microscope level, the cytotoxic changes produced by the venom of this spider.Cultures of human epithelial carcinoma cells, strain HeLa, were initiated on sterile, carbon coated coverslips contained in Leighton tubes. Each culture was seeded with approximately 1x105 cells contained in 1.5 ml of a modified Eagle's minimum essential growth medium prepared in Hank's balanced salt solution. Cultures were incubated at 36° C. for three days prior to the addition of venom. The venom was collected from female brown recluse spiders and diluted in sterile saline. Protein determinations on the venom-were made according to the spectrophotometric method of Waddell (1956). Approximately 10 μg venom protein per ml of fresh medium was added to each culture after discarding the old growth medium. Control cultures were treated similarly, except that no venom was added. All cultures were reincubated at 36° C.

Use of Uranium-Labeled Antibodies for Electron Microscopic Localization of Various Antigens in Mammalian Cells

1967 ◽  
Vol 25 ◽  
pp. 136-137
Author(s):  
J. P. Petrali ◽  
E. J. Donati ◽  
L. A. Sternberger
Keyword(s):  
Endoplasmic Reticulum ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Specific Antiserum ◽  
Electron Microscopic ◽  
E Coli ◽  
Cytoplasmic Membranes ◽  
Viral Progeny ◽  
Ultrathin Sections ◽  
Electron Microscopic Localization

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

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