Enzymes are powerful tools for protein labelling due to their specificity and mild reaction conditions. Many protocols, however, are restricted to modifications at protein termini, rely on non-peptidic metabolites or require large recognition domains. Here we report a chemoenzymatic method, which we call lysine acylation using conjugating enzymes (LACE), to site-specifically modify folded proteins at internal lysine residues. LACE relies on a minimal genetically encoded tag (four residues) recognized by the E2 small ubiquitin-like modifier-conjugating enzyme Ubc9, and peptide or protein thioesters. Together, this approach obviates the need for E1 and E3 enzymes, enabling isopeptide formation with just Ubc9 in a programmable manner. We demonstrate the utility of LACE by the site-specific attachment of biochemical probes, one-pot dual-labelling in combination with sortase, and the conjugation of wild-type ubiquitin and ISG15 to recombinant proteins.

由于其特异性和温和的反应条件，酶是蛋白质标记的有力工具。然而，许多协议仅限于蛋白质末端的修改，依赖于非肽代谢物或需要大的识别域。在这里，我们报告了一种化学酶法，我们将其称为使用结合酶 (LACE) 的赖氨酸酰化，以在内部赖氨酸残基处对折叠蛋白质进行定点修饰。LACE 依赖于由 E2 小泛素样修饰符缀合酶 Ubc9 和肽或蛋白质硫酯识别的最小基因编码标签（四个残基）。总之，这种方法不需要 E1 和 E3 酶，只需 Ubc9 即可以可编程方式形成异肽。我们通过生化探针的位点特异性附着来证明 LACE 的效用，