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

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.

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Engineering an acetyllysine reader with photocrosslinking amino acid for interactome profiling

Chemical Communications ◽

10.1039/d1cc04611j ◽

2021 ◽

Author(s):

Babu Sudhamalla ◽

Anirban Roy ◽

Soumen Barman ◽

Jyotirmayee Padhan

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Protein Interactions ◽

Unnatural Amino Acids ◽

Protein Protein Interactions ◽

Site Specific ◽

Powerful Approach

The site-specific installation of light-activable crosslinker unnatural amino acids offers a powerful approach to trap transient protein-protein interactions both in vitro and in vivo. Herein, we engineer a bromodomain to...

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

RNA ◽

10.1261/rna.2024810 ◽

2010 ◽

Vol 16 (8) ◽

pp. 1660-1672 ◽

Cited By ~ 16

Author(s):

J. Gubbens ◽

S. J. Kim ◽

Z. Yang ◽

A. E. Johnson ◽

W. R. Skach

Keyword(s):

Amino Acids ◽

Nonnatural Amino Acids ◽

Amber Suppressor

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The potentials of the in vitro protein biosynthesis system

Journal of Biotechnology ◽

10.1016/0168-1656(95)00005-b ◽

1995 ◽

Vol 41 (2-3) ◽

pp. 81-90 ◽

Cited By ~ 31

Author(s):

Wolfgang Stiege ◽

Volker A. Erdmann

Keyword(s):

Protein Biosynthesis ◽

Vitro Protein

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Expansion of protein biosynthesis system including nonnatural amino acids

2006 IEEE International Symposium on MicroNanoMechanical and Human Science ◽

10.1109/mhs.2006.320251 ◽

2006 ◽

Author(s):

Takashi Ohtsuki ◽

Yoshio Doi ◽

Taishi Manabe ◽

Masahiko Sisido

Keyword(s):

Amino Acids ◽

Protein Biosynthesis ◽

Nonnatural Amino Acids

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

Canadian Journal of Biochemistry ◽

10.1139/o69-189 ◽

1969 ◽

Vol 47 (12) ◽

pp. 1179-1186 ◽

Cited By ~ 2

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.

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