Efficient Incorporation of Nonnatural Amino Acids with Large Aromatic Groups into Streptavidin in In Vitro Protein Synthesizing Systems

1999 ◽  
Vol 121 (1) ◽  
pp. 34-40 ◽  
Author(s):  
Takahiro Hohsaka ◽  
Daisuke Kajihara ◽  
Yuki Ashizuka ◽  
Hiroshi Murakami ◽  
Masahiko Sisido
Keyword(s):  
Amino Acids ◽  
Nonnatural Amino Acids ◽  
Vitro Protein

Site-specific incorporation of photofunctional nonnatural amino acids into a polypeptide through in vitro protein biosynthesis

FEBS Letters ◽  
1994 ◽  
Vol 344 (2-3) ◽  
pp. 171-174 ◽  
Author(s):  
Takahiro Hohsaka ◽  
Ken Sato ◽  
Masahiko Sisido ◽  
Kazuyuki Takai ◽  
Shigeyuki Yokoyama
Keyword(s):  
Amino Acids ◽  
Protein Biosynthesis ◽  
Nonnatural Amino Acids ◽  
Site Specific ◽  
Vitro Protein

scholarly journals Chemical aminoacylation of tRNAs with fluorinated amino acids for in vitro protein mutagenesis

2010 ◽  
Vol 6 ◽  
Author(s):  
Shijie Ye ◽  
Allison Ann Berger ◽  
Dominique Petzold ◽  
Oliver Reimann ◽  
Benjamin Matt ◽  
...  
Keyword(s):  
Amino Acids ◽  
Translation System ◽  
Biologically Relevant ◽  
Fluorinated Amino Acids ◽  
Free Translation ◽  
Trna Species ◽  
A Cell ◽  
Protein Environment ◽  
Vitro Protein

This article describes the chemical aminoacylation of the yeast phenylalanine suppressor tRNA with a series of amino acids bearing fluorinated side chains via the hybrid dinucleotide pdCpA and ligation to the corresponding truncated tRNA species. Aminoacyl-tRNAs can be used to synthesize biologically relevant proteins which contain fluorinated amino acids at specific sites by means of a cell-free translation system. Such engineered proteins are expected to contribute to our understanding of discrete fluorines’ interaction with canonical amino acids in a native protein environment and to enable the design of fluorinated proteins with arbitrary desired properties.

scholarly journals In vitro incorporation of nonnatural amino acids into protein using tRNACys-derived opal, ochre, and amber suppressor tRNAs

RNA ◽  
2010 ◽  
Vol 16 (8) ◽  
pp. 1660-1672 ◽  
Author(s):  
J. Gubbens ◽  
S. J. Kim ◽  
Z. Yang ◽  
A. E. Johnson ◽  
W. R. Skach
Keyword(s):  
Amino Acids ◽  
Nonnatural Amino Acids ◽  
Amber Suppressor

In vitro protein synthesis directed by R17 viral ribonucleic acid. II. On the fate of mRNA in the cell-free system

1969 ◽  
Vol 47 (12) ◽  
pp. 1179-1186 ◽  
Author(s):  
Satomi J. Igarashi
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid Incorporation ◽  
Free System ◽  
Cell Free System ◽  
E Coli ◽  
Acid Incorporation ◽  
Binding Factor ◽  
Vitro Protein

In the crude E. coli B cell-free system, mRNA was hydrolyzed by contaminating nuclease activities before significant polymerization of amino acids took place. Ribosomes appeared to be one of the sources of nuclease. A modified high-salt washing procedure was developed to remove nuclease from ribosomes. RNase-free ribosomes thus obtained appeared to be inactive in poly-U-directed phenylalanine incorporation, unless poly-U binding factor was added to the system. R17 RNA could not direct amino acid incorporation in the presence of RNase-free ribosomes because binding of intact R17 RNA to ribosomes did not take place even in the presence of poly-U binding factor.

scholarly journals Amino Acids 785, 787 of the Na+/H+ Exchanger Cytoplasmic Tail Modulate Protein Activity and Tail Conformation

2021 ◽  
Vol 22 (21) ◽  
pp. 11349
Author(s):  
Xiuju Li ◽  
Tommy Tu ◽  
Sicheng Quan ◽  
Francisco J. Quintero ◽  
Richard Fahlman ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Apparent Molecular Weight ◽  
Slight Reduction ◽  
Membrane Domain ◽  
Protein Activity ◽  
Extracellular Sodium ◽  
Vitro Protein

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a plasma membrane protein ubiquitously present in humans. It regulates intracellular pH by removing an intracellular proton in exchange for an extracellular sodium. It consists of a 500 amino acid membrane domain plus a 315 amino acid, regulatory cytosolic tail. Here, we investigated the effect of mutation of two amino acids of the regulatory tail, Ser785 and Ser787, that were similar in location and context to two amino acids of the Arabidopsis Na+/H+ exchanger SOS1. Mutation of these two amino acids to either Ala or phosphomimetic Glu did not affect surface targeting but led to a slight reduction in the level of protein expressed. The activity of the NHE1 protein was reduced in the phosphomimetic mutations and the effect was due to a decrease in Vmax activity. The Ser to Glu mutations also caused a change in the apparent molecular weight of both the full-length protein and of the cytosolic tail of NHE1. A conformational change in this region was indicated by differential trypsin sensitivity. We also found that a peptide containing amino acids 783–790 bound to several more proximal regions of the NHE1 tail in in vitro protein interaction experiments. The results are the first characterization of these two amino acids and show that they have significant effects on enzyme kinetics and the structure of the NHE1 protein.

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