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Publications 2010 - 2012

Conjugation of Proteins by Installing BIO-Orthogonally Reactive Groups at Their N-Termini
Citation key 098.2012.soundrarajan
Author Soundrarajan, N. and Sokalingam, S. and Raghunathan, G. and Budisa, N. and Paik, H. J. and Yoo, T.H. and Lee S. G.
Pages e46741
Year 2012
DOI 10.1371/journal.pone.0046741
Journal PLoSONE
Volume 2012
Number 7
Abstract N-terminal site-specific modification of a protein has many advantages over methods targeting internal positions, but it is not easy to install reactive groups onto a protein in an N-terminal specific manner. We here report a strategy to incorporate amino acid analogues specifically in the N-terminus of a protein in vivo and demonstrate it by preparing green fluorescent protein (GFP) having bio-orthogonally reactive groups at its N-terminus. In the first step, GFP was engineered to be a foldable, internal methionine-free sequence via the semi-rational mutagenesis of five internal methionine residues and the introduction of mutations for GFP folding enhancement. In the second step, the N-terminus of the engineered protein was modified in vivo with bio-orthogonally functional groups by reassigning functional methionine surrogates such as L-homopropargylglycine and L-azidohomoalanine into the first methionine codon of the engineered internal methionine-free GFP. The N-terminal specific incorporation of unnatural amino acids was confirmed by ESI-MS analysis and the incorporation did not affect significantly the specific activity, refolding rate and folding robustness of the protein. The two proteins which have alkyne or azide groups at their N-termini were conjugated each other by bio-orthogonal Cu(I)-catalyzed click chemistry. The strategy used in this study is expected to facilitate bio-conjugation applications of proteins such as N-terminal specific glycosylation, labeling of fluorescent dyes, and immobilization on solid surfaces.
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