scholarly journals Development of a Novel Q-body Using an In Vivo Site-Specific Unnatural Amino Acid Incorporation System

Sensors ◽  
2018 ◽  
Vol 18 (8) ◽  
pp. 2519
Author(s):  
Yoichi Kurumida ◽  
Nobuhiro Hayashi
Keyword(s):  
Amino Acid ◽  
Fluorescence Intensity ◽  
Unnatural Amino Acid ◽  
Fluorescent Dye ◽  
Amino Acid Residues ◽  
Amino Acid Incorporation ◽  
System A ◽  
Acid Incorporation ◽  
Tryptophan Residues

A Q-body capable of detecting target molecules in solutions could serve as a simple molecular detection tool. The position of the fluorescent dye in a Q-body affects sensitivity and therefore must be optimized. This report describes the development of Nef Q-bodies that recognize Nef protein, one of the human immunodeficiency virus (HIV)’s gene products, in which fluorescent dye molecules were placed at various positions using an in vivo unnatural amino acid incorporation system. A maximum change in fluorescence intensity of 2-fold was observed after optimization of the dye position. During the process, some tryptophan residues of the antibody were found to quench the fluorescence. Moreover, analysis of the epitope indicated that some amino acid residues of the antigen located near the epitope affected the fluorescence intensity.

scholarly journals Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Amino Acids ◽  
2020 ◽  
Author(s):  
Thomas L. Williams ◽  
Debra J. Iskandar ◽  
Alexander R. Nödling ◽  
Yurong Tan ◽  
Louis Y. P. Luk ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Genetic Code ◽  
Unnatural Amino Acids ◽  
Trna Synthetase ◽  
Unnatural Amino Acid ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Genetic Code Expansion ◽  
Incorporation Efficiency

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

Reduced and S-carboxymethylated human growth hormone: a probe for diabetogenic action

1984 ◽  
Vol 247 (5) ◽  
pp. E639-E644
Author(s):  
C. M. Cameron ◽  
J. L. Kostyo ◽  
J. A. Rillema ◽  
S. E. Gennick
Keyword(s):  
Growth Hormone ◽  
Amino Acid ◽  
Human Growth Hormone ◽  
Human Growth ◽  
Mouse Mammary Gland ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Hypophysectomized Rats

The biological activity profile of reduced and S-carboxymethylated human growth hormone (RCM-hGH) was determined to establish its suitability for study of the diabetogenic property of hGH. RCM-hGH was found to have greatly attenuated in vivo growth-promoting activity in the 9-day weight-gain test in hypophysectomized rats (approximately 1%) and to have a similar low order of in vitro activity in stimulating amino acid incorporation into the protein of the isolated rat diaphragm. RCM-hGH also only had approximately 1% of the in vitro insulin-like activity of the native hormone on isolated adipose tissue from hypophysectomized rats. In contrast, RCM-hGH retained substantial in vivo diabetogenic activity in the ob/ob mouse, appearing to have approximately 50% of the activity of the native hormone. RCM-hGH was also found to retain significant, although attenuated (25%), in vitro lactogenic activity when tested for the ability to stimulate amino acid incorporation into a casein-rich protein fraction in mouse mammary gland explants. Because RCM-hGH exhibits a high degree of diabetogenic activity, although lacking significant anabolic or insulin-like activities, it will be useful as a "monovalent" probe for the study of the molecular mechanism of the diabetogenic action of GH.

Nicotinic receptor binding site probed with unnatural amino acid incorporation in intact cells

Science ◽  
1995 ◽  
Vol 268 (5209) ◽  
pp. 439-442 ◽  
Author(s):  
M. Nowak ◽  
P. Kearney ◽  
Sampson ◽  
M. Saks ◽  
C. Labarca ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Receptor Binding ◽  
Nicotinic Receptor ◽  
Unnatural Amino Acid ◽  
Amino Acid Incorporation ◽  
Receptor Binding Site ◽  
Intact Cells ◽  
Acid Incorporation

Effect of Cycloheximide on In Vitro and In Vivo Protein Synthesis by Certain Tissues of Rats and Pigeons with Special Reference to Muscle

1973 ◽  
Vol 51 (12) ◽  
pp. 933-941 ◽  
Author(s):  
Njanoor Narayanan ◽  
Jacob Eapen
Keyword(s):  
Amino Acids ◽  
Body Weight ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Amino Acid Incorporation ◽  
Contrast Administration ◽  
Acid Incorporation ◽  
Tissue Homogenates

The effect of cycloheximide in vitro and in vivo on the incorporation of labelled amino acids into protein by muscles, liver, kidneys, and brain of rats and pigeons was studied. In vitro incorporation of amino acids into protein by muscle microsomes, myofibrils, and myofibrillar ribosomes was not affected by cycloheximide. In contrast, administration of the antibiotic into intact animals at a concentration of 1 mg/kg body weight resulted in considerable inhibition of amino acid incorporation into protein by muscles, liver, kidneys, and brain. This inhibition was observed in all the subcellular fractions of these tissues during a period of 10–40 min after the administration of the precursor. Tissue homogenates derived from in vivo cycloheximide-treated animals did not show significant alteration in in vitro amino acid incorporation with the exception of brain, which showed a small but significant enhancement.

scholarly journals Biotin-mediated protein biosynthesis

1974 ◽  
Vol 140 (3) ◽  
pp. 549-556 ◽  
Author(s):  
R. L. Boeckx ◽  
K. Dakshinamurti
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Rat Liver ◽  
Intestinal Mucosa ◽  
Protein Biosynthesis ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Stimulation Of

The effect of administration of biotin to biotin-deficient rats on protein biosynthesis was studied. Biotin treatment resulted in stimulation by more than twofold of amino acid incorporation into protein, both in vivo and in vitro in rat liver, pancreas, intestinal mucosa and skin. Analysis of the products of amino acid incorporation into liver proteins in vivo and in vitro indicated that the synthesis of some proteins was stimulated more than twofold, but others were not stimulated at all. This indicates a specificity in the stimulation of protein synthesis mediated by biotin.

Incorporation of C14-amino acids into protein of isolated diaphragms: effect of epinephrine and norepinephrine

1960 ◽  
Vol 198 (1) ◽  
pp. 54-56 ◽  
Author(s):  
Ira G. Wool
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Glucose Concentration ◽  
Muscle Protein ◽  
Effective Concentration ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Minimal Effective Concentration

When diaphragms isolated from normal rats were incubated with a C14-amino acid the addition of epinephrine or norepinephrine decreased incorporation of C14 into muscle protein. The inhibition occurred whether epinephrine was added in vitro or administered in vivo. The minimal effective concentration of epinephrine in vitro was 0.1 µg/ml. When the glucose concentration in the medium was raised to 300 mg % or more the epinephrine induced inhibition of amino acid incorporation into muscle protein was no longer observed.

scholarly journals Agranular membranes in free polysome preparations and their possible interference in studies of protein biosynthesis

1969 ◽  
Vol 112 (3) ◽  
pp. 269-274 ◽  
Author(s):  
C. N. Murty ◽  
T. Hallinan
Keyword(s):  
Amino Acid ◽  
Functional Capacity ◽  
Initial Rate ◽  
Protein Biosynthesis ◽  
Amino Acid Incorporation ◽  
Electron Microscopic ◽  
Acid Incorporation ◽  
Microscopic Studies ◽  
Purification Methods

1. Phospholipid-rich membranous contaminants are present in free polysomes from rat liver isolated on discontinuous sucrose gradients. 2. Electron-microscopic studies indicate that the membranous contaminants are mainly agranular with very occasional granular membranes. This is confirmed by the study of their sedimentation behaviour and their initial rate of labelling with radioactive glucosamine in vivo. 3. Conventional ribosome-purification methods fail to remove the contaminants, whereas deoxycholate effectively solubilizes the membranous contaminants with little breakdown of polysomes. 4. Amino acid-incorporation studies show that these membranous contaminants may seriously interfere in assessment of the functional capacity of free polysomes in protein biosynthesis in vivo.

Sign in / Sign up

Export Citation Format

Share Document