Transpeptidase-catalyzed multi-fragment assemblies of peptides and proteins

转肽酶是蛋白质化学的有力工具，它使合成肽和重组蛋白的化学酶连接成为可能。本提案的中心目标是开发多种肽和蛋白质的化学酶连接的新方法。我们最近报道了通过排序酶介导的连接（SML）使用可切换的连接位点组装多肽。转肽酶分类酶A识别LPxTG分类基序，在苏氨酸残基上切割序列，并将酶结合的中间体连接到具有n端甘氨酸亲核试剂的肽上。我们发现亮氨酸残基可以被二硫化物保护的半胱氨酸取代，从而允许SML。在二硫还原后，分选基序在连接产物中失活。当与光笼型亲核试剂结合时，多个多肽可以通过SML连接。在这里，我们计划通过将活化的二硫化物与蛋白酶活化的亲核试剂一起引入重组蛋白的分选基序中，在蛋白质水平上建立可切换的连接位点。这种方法的实用性应该通过组装乙酰赖氨酸结合模块抑制剂的报告基因来证明。在第二个项目中，我们计划在排序基序中建立以脯氨酸残基为重点的替代开关位点。已知该残基在排序基序的构象中引入扭结，这对于排序酶结合是必不可少的。通过引入具有酸或光不稳定主链修饰的氨基酸，我们计划在SML期间保持扭结构象，并在酸处理或紫外线照射后转换为延长的非活性构象。在最后一个项目中，我们计划将分选酶和植物转肽酶丁酶1结合在一个正交三片段连接方案中，实现丁酶的高效重组表达。考虑到酶的内质网定位，我们计划评估和优化丁烯酶在哺乳动物Expi293细胞和利什曼原虫中的表达，这两种细胞都已经优化了重组蛋白的内质网靶向。应该建立乙酰赖氨酸结合模块抑制剂的报告结构的一锅连接，证明这种正交连接方案的实用性。

Transpeptidases are powerful tools of protein chemistry by enabling chemoenzymatic ligations of synthetic peptides and recombinant proteins. The central aim of this proposal is the development of new methods for chemoenzymatic ligations of multiple peptides and proteins. We have recently reported multi-peptide assemblies by sortase-mediated ligation (SML) using switchable ligation sites. The transpeptidase sortase A recognizes an LPxTG sorting motif, cleaves the sequence at the threonine residue and ligates the enzyme-bound intermediate to a peptide with N-terminal glycine nucleophile. We showed that the leucine residue can be replaced by a disulfide-protected cysteine allowing SML. Upon disulfide reduction the sorting motif is inactivated in the ligation product. When combined with a photo-caged nucleophile multiple peptides can be ligated by SML. Here we plan to establish switchable ligation sites on protein level by introducing activating disulfides into sorting motifs of recombinant proteins together with protease-activatable nucleophiles. The utility of this approach should be demonstrated by assembling a reporter for inhibitors of acetyllysine binding modules. In a second project, we plan to establish alternative switch sites in the sorting motif focusing on the proline residue. This residue is known to introduce a kink into the conformation of the sorting motif which is essential for sortase binding. By introducing amino acids with acid- or photo-labile backbone modifications, we plan to maintain the kinked conformation during SML and switch to an extended inactive conformation afterwards by acid treatment or UV irradiation. In the last project, we plan to combine sortase and plant transpeptidase butelase 1 in an orthogonal three-fragment ligation scheme by enabling efficient recombinant expression of butelase. Taking the ER localization of the enzyme into account, we plan to evaluate and optimize butelase expression in mammalian Expi293 cells and Leishmania tarentolae, both of which have been optimized for ER targeting of recombinant proteins. A one-pot ligation of the reporter construct for acetyllysine binding module inhibitors should be established, demonstrating utility of this orthogonal ligation scheme.