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• W1964575772 endingPage "e46741" @default.
• W1964575772 startingPage "e46741" @default.
• W1964575772 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." @default.
• W1964575772 created "2016-06-24" @default.
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• W1964575772 date "2012-10-08" @default.
• W1964575772 modified "2023-10-03" @default.
• W1964575772 title "Conjugation of Proteins by Installing BIO-Orthogonally Reactive Groups at Their N-Termini" @default.
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• W1964575772 doi "https://doi.org/10.1371/journal.pone.0046741" @default.
• W1964575772 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3466299" @default.
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• W1964575772 hasPublicationYear "2012" @default.
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