Efficient N-terminal labeling of proteins by use of sortase.
Efficient N-terminal labeling of proteins by use of sortase.
复制标题
DOI：
10.1002/anie.201204538
复制
发表时间：
2012-09-10
期刊：
影响因子：
--
通讯作者：
Turnbull, W Bruce
中科院分区：
文献类型：
--
作者：
Williamson, Daniel J;Fascione, Martin A;Turnbull, W Bruce
Protein labeling is a pivotal technique in molecular and cell biology. Strategies include derivatization of cysteine residues,[1] labeling lysine or N-terminal amino groups with activated esters, periodate or PLP-mediated oxidation of the N-terminus for oxime ligation,[2] and native-chemical ligation.[3] Each of these methods has its own associated challenges: Selective labeling of a single cysteine residue frequently requires rounds of site-directed mutagenesis to introduce the labeling site and/or remove other cysteine residues, and selective labeling of the N-terminal amino group requires careful control of pH to ensure lysine residues are not also modified.[4] Other modern methods for chemoselective labeling often require the introduction of specific recognition sequences [5] or nonnatural amino acids into the protein to be labeled.[6] In most cases, a substantial excess of the labeling reagent is necessary to ensure complete conversion to the product. We report a method for chemoselective N-terminal labeling of recombinant proteins in quantitative yield using depsipeptide substrates for the transpeptidase sortase A. The method does not require engineering of the protein sequence beyond that typically used in contemporary recombinant protein purification strategies. Unlike previous approaches,[7] the method requires only a single N-terminal glycine residue in a sterically unhindered position, a minimal excess of the labeling reagent and substoichiometric quantities of transpeptidase. Sortase A (SrtA) catalyzes the reversible attachment of virulence factors to the cell walls of Gram positive bacteria by C-terminal modification of proteins at an LPXTG recognition sequence.[8] The enzyme catalyzes the covalent attachment of the LPXT motif to a cysteine residue in the catalytic site to form a thioester intermediate. An N-terminal oligoglycine motif in the peptidoglycan can then react with this intermediate to covalently attach the substrate to the cell wall.SrtA has been exploited extensively for the C-terminal modification of proteins.[7b, 9] However, this method has certain constraints: the LPXTG sequence must be engineered into the protein and excess nucleophilic labeling reagent is required to push the equilibrium toward formation of product as the transpeptidase reaction is reversible (Scheme 1a).
