Position-specific incorporation of biotinylated non-natural amino acids into a protein in a cell-free translation system

doi:10.1016/j.bbrc.2007.07.099

Biochemical and Biophysical Research Communications

Volume 361, Issue 3, 28 September 2007, Pages 794-799

https://doi.org/10.1016/j.bbrc.2007.07.099 Get rights and content

Abstract

Biotinylation is useful for the detection, purification and immobilization of proteins. It is performed by chemical modification, although position-specific and quantitative biotinylation is rarely achieved. We developed a position-specific biotinylation method using biotinylated non-natural amino acids. We showed that biotinylated p-aminophenylalanine derivatives were incorporated into a protein more efficiently than biotinylated lysine derivatives in a cell-free translation system. In addition, the biotinylated p-aminophenylalanines overcame the serious position-dependency observed for biotinylated lysines. The present method will be useful for detection and purification of proteins along with comprehensive exploration of surface-exposed residues and oriented immobilization of proteins.

Section snippets

Materials and methods

Synthesis of biotinylated aminophenylalanyl-pdCpA. To p-aminophenylalanyl-pdCpA [13] (0.44 μmol) and N-hydroxysuccinimide biotin (2 μmol, Sigma) in DMSO (400 μl), 1 M aqueous pyridine–HCl (pH 5.0, 400 μl) was added and incubated at 37 °C for 8 h. The desired p-(biotinylamino)phenylalanyl-pdCpA was purified by a preparative reverse-phase HPLC (XBridge C18 (Waters), 5 μm, 10× 50 mm), flow rate 3.0 ml/min with a linear gradient of 0–100% methanol in 0.38% formic acid, over 15 min. The product was identified

Incorporation of biotinylated amino acids

Biotinylated aminoacyl-tRNAs were prepared by the chemical aminoacylation method [15], [16]. Dinucleotide pdCpA aminoacylated with BioAF or BioXAF was chemically synthesized by the reaction of aminophenylalanyl-pdCpA with biotin succinimide ester or biotinamidocaproyl succinimide ester in aqueous pyridine–HCl buffer (pH 5). At pH 5, the p-amino group was partially deprotonated and selectively reacted with the succinimide ester. BioLys- or BioXLys-pdCpA were synthesized by the reaction of

Acknowledgments

This work was partially supported by the Industrial Technology Research Grant Program (00A03018a) from the New Energy and Industrial Technology Development Organization of Japan, and Grant-in-Aid for Young Scientists (17685017) from the Ministry of Education, Science, Sports and Culture, Japan.

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Position-specific incorporation of biotinylated non-natural amino acids into a protein in a cell-free translation system

Abstract

Section snippets

Materials and methods

Incorporation of biotinylated amino acids

Acknowledgments

Methods Enzymol.

Methods Enzymol.

J. Biosci. Bioeng.

Chem. Biol.

FEBS Lett.

Anal. Biochem.

J. Biol. Chem.

Intein-mediated biotinylation of proteins and its application in a protein microarray

J. Am. Chem. Soc.