转肽酶Sortase A催化的新反应及其在蛋白质修饰中的应用

本项目拟发展转肽酶Sortase A催化的新型多肽/蛋白质末端氨基酸转酰肼化反应并将其应用于蛋白质的定点标记、修饰及半合成。具体课题设计及拟实现目标主要包括：⑴ 以C端含有Sort A识别序列（LPXTG等）的多肽为模型，在外加Sort A与亲核试剂（水合肼）的条件下得到C端转化为酰肼末端的目标多肽并筛选最佳转化条件，实现末端多肽酰肼的酶促合成；⑵ 将多肽酰肼的酶促合成应用于功能蛋白，并实现蛋白质的C端标记、修饰及半合成；⑶ 研究Sort A催化的多肽/蛋白酰肼的一步法酶促合成，发现转肽酶硫脂中间体能被取代水合肼肼解，实现一步法的标记和修饰；⑷ 基于上述方法实现在生理条件下蛋白质药物的一步法修饰和标记，在保持其药用活性的前提下改善水溶性和酶稳定性，最终达到提高其医药学价值的应用目标。

In the proposed project, we plan to develop a new peptide/protein C-terminal amio acids trans-hydrazidiase reaction which is catalyzed by transpeptidase Sortase A. This reaction could be further applicated in site-specific protein labeling, modification and semi-synthesis. We design to realize series of objective including: ⑴ firstly, Sortase A targeted substrate peptide bearing a "sorting motif" was used as model to realize C terminal peptide hydrazide synthesis under the catalysis of Sortase A in combination with hydrazine. The efficiency of this reaction should be optimized subsequently; ⑵ secondly, Sortase A catalysis peptide hydrazide synthsis should be further expanded to bio-function protein. We aim to realize C terminal protein labeling, modification and semi-synthesis via this new bioorthogonal reaction; ⑶ thirdly, to realize one-step protein labeling and modificaiton, one-step Sortase A catalyzed peptide/protein hydrazide synthesis reaction should be studied by the hydrazinolysis of a Sortase A formed thioacyl intermediate; ⑷ finally, based on aforementioned new reaction, we aim to realize one-step site-spectific therapeutic protein modification in physiological condition. Thus, the medicinal value of therapeutic protein should be increased by substantially improved solubility and stability, increased the half-life of the protein in vivo as well as masking potentially immunogenic epitopes.