Peptide Logic Circuits Based on Chemoenzymatic Ligation for Programmable Cell Apoptosis

A novel and versatile peptide‐based bio‐logic system capable of regulating cell function is developed using sortase A (SrtA), a peptide ligation enzyme, as a generic processor. By modular peptide design, we demonstrate that mammalian cells apoptosis can be programmed by peptide‐based logic operations, including binary and combination gates (AND, INHIBIT, OR, and AND‐INHIBIT), and a complex sequential logic circuit (multi‐input keypad lock). Moreover, a proof‐of‐concept peptide regulatory circuit was developed to analyze the expression profile of cell‐secreted protein biomarkers and trigger cancer‐cell‐specific apoptosis.     

Sortase-Mediated Transpeptidation for Site-Specific Modification of Peptides,Glycopeptides, and Proteins

Sortases are a family of transpeptidases found in gram-positive bacteria responsible for covalent anchoring of cell surface proteins to bacterial cell walls. It has been discovered that sortase A (SrtA) of Staphylococcus aureus origin is rather promiscuous and can accept various molecules as substrates. As a result, SrtA has been widely used to ligate peptides and proteins with a variety of nucleophiles, and the ligation products are useful for research in chemical biology, proteomics, biomedicine, etc. This review summarizes the recent applications of SrtA with special emphasis on SrtA-catalyzed ligation of carbohydrates with peptides and proteins.     

转肽酶Sortase A在蛋白质修饰中的应用

近年来,发展蛋白质与多肽的化学选择性连接与修饰方法已成为化学生物学的研究热点。这类方法可以在很大程度上弥补基因工程技术无法制备翻译后修饰蛋白与人工改造蛋白的缺陷,得到目前无法通过生物表达的功能蛋白。20世纪90年代末,人们从金黄色葡萄球菌中分离得到转肽酶Sortase A,它能够选择性识别特异性多肽序列LPXTG,并在特定位点切断氨基酸的肽键进而将其与一个新的肽链连接。该特性使Sortase A有望成为一种高效、通用的蛋白质修饰的工具。与传统的化学合成相比,Sortase催化的化学半合成方法,可以较好的解决化学合成蛋白的尺寸问题。本文就近年来Sortase A在蛋白质修饰及合成中的研究进展进行简要综述。     

Proximity‐Based Sortase‐Mediated Ligation

Protein bioconjugation has been a crucial tool for studying biological processes and developing therapeutics. Sortase A (SrtA), a bacterial transpeptidase, has become widely used for its ability to site‐specifically label proteins with diverse functional moieties, but a significant limitation is its poor reaction kinetics. In this work, we address this by developing proximity‐based sortase‐mediated ligation (PBSL), which improves the ligation efficiency to over 95 % by linking the target protein to SrtA using the SpyTag–SpyCatcher peptide–protein pair. By expressing the target protein with SpyTag C‐terminal to the SrtA recognition motif, it can be covalently captured by an immobilized SpyCatcher–SrtA fusion protein during purification. Following the ligation reaction, SpyTag is cleaved off, rendering PBSL traceless, and only the labeled protein is released, simplifying target protein purification and labeling to a single step.     

A Versatile Approach for the Site‐Specific Modification of Recombinant Antibodies Using a Combination of Enzyme‐Mediated Bioconjugation and Click Chemistry

A unique two‐step modular system for site‐specific antibody modification and conjugation is reported. The first step of this approach uses enzymatic bioconjugation with the transpeptidase Sortase A for incorporation of strained cyclooctyne functional groups. The second step of this modular approach involves the azide–alkyne cycloaddition click reaction. The versatility of the two‐step approach has been exemplified by the selective incorporation of fluorescent dyes and a positron‐emitting copper‐64 radiotracer for fluorescence and positron‐emission tomography imaging of activated platelets, platelet aggregates, and thrombi, respectively. This flexible and versatile approach could be readily adapted to incorporate a large array of tailor‐made functional groups using reliable click chemistry whilst preserving the activity of the antibody or other sensitive biological macromolecules.