Zhang Wenbin research group of Peking University develops protein stapler enzymes to achieve efficient peptide peptide coupling reaction

Efficient peptide peptide coupling reactions are of great significance in the preparation of protein drug conjugates, the regulation of protein topologies, biological imaging, chemical biology and other fields, and have attracted much attention due to their gene coding properties At present, there are not many reported methods of this kind of connection, among which sortase or butelase-1-mediated coupling reaction has good substrate sequence specificity, and protein connection method mediated by peptide (I ntein) can achieve high-efficiency main chain connection with gene coding, while transglutaminase (transglutaminase) can achieve high-efficiency main chain connection ）It can mediate nonspecific side chain isopeptide coupling In recent years, the rapid development of peptide protein reaction pairs has provided a new opportunity for the development of such tools The peptide peptide coupling system mediated by spy ligase and snoop ligase has been reported, but its efficiency still needs to be improved, and its application prospect in cells needs to be explored Recently, Zhang Wenbin's research group of Peking University has developed a unique spy stapler with disordered structure, which realizes the efficient coupling of spy tag and bdtag in and out of cells Based on the crystal structure analysis of spy catcher complex, the spy catcher was separated from the second ring region of its structure, and the Peking University tag and spy stapler enzyme were obtained The results show that spy stapler enzyme can promote the formation of isopeptide bond between spy tag and Peking University tag in both extracellular and intracellular environment, and the yield can reach more than 80% under mild conditions The coupling method can be used in protein topology engineering to prepare four arm star shaped elastin and cyclic dihydrofolate reductase by extracellular reaction It is worth noting that spy stapler enzyme can also work efficiently in cells As long as we co express linear reactive dihydrofolate reductase and spy stapler enzyme, we can directly realize cyclization in situ and quantitatively in cells (Figure 1) Generally, enzymes need stable and orderly folding structure to achieve their functions Spy stapler enzymes are in sharp contrast Circular dichroism and nuclear magnetic resonance spectroscopy confirmed that the enzyme of spy stapler was completely disordered in the solution and did not have a stable folding structure Only when spy tag and Peking University TAG exist at the same time, can it form a stable secondary structure Because its activity does not depend on the pre-existing ordered structure, spy stapler enzyme can withstand heating, repeated freezing and thawing, chemical denaturation and other treatments without losing its activity The high reaction efficiency and unique stability of spy stapler system provide a new tool for peptide peptide coupling and protein topology engineering Fig 1 (a) spy stapler enzyme can mediate the coupling between polypeptide tags in the flexible chain to form a four armed star shaped elastin; (b) co expression of reactive dihydrofolate reductase and spy stapler enzyme in the cell can realize the in-situ cyclization of protein in the cell (source: J am Chem SOC.) the study was recently published online in J am Chem SOC (DOI: 10.1021 / JACS 8b08250) Wu Xialing and Liu Yajie, Ph.D students of the school of chemistry and molecular engineering of Peking University, are the co first authors of the paper, and Zhang Wenbin, a researcher, is the corresponding author Professor Sun Fei of the Hong Kong University of science and technology also participated in the study This work was supported by the National Natural Science Foundation of China, 863 program, youth thousand talents program, interdisciplinary medical seed fund of Peking University and the open project of National Key Laboratory of supramolecular structure and materials of Jilin University.