Transpeptidase-catalyzed multi-fragment assemblies of peptides and proteins

转肽酶是蛋白质化学的有力工具，它能使合成肽和重组蛋白质发生化学酶促连接。该提案的中心目标是开发用于多个肽和蛋白质的化学酶促连接的新方法。我们最近报道了通过分选酶介导的连接（SML）使用可切换的连接位点的多肽组件。转肽酶分选酶A识别LPxTG分选基序，在苏氨酸残基处切割序列，并将酶结合的中间体连接到具有N-末端甘氨酸亲核体的肽。我们表明，亮氨酸残基可以被二硫键保护的半胱氨酸取代，从而允许SML。二硫键还原后，连接产物中的分选基序失活。当与光笼化亲核试剂组合时，多个肽可以通过SML连接。在这里，我们计划建立蛋白质水平上的可切换的连接位点，通过引入活化二硫键到重组蛋白的分选基序与蛋白酶激活的亲核试剂。这种方法的效用应证明组装乙酰赖氨酸结合模块的抑制剂的报告。在第二个项目中，我们计划建立替代开关网站的排序基序集中在脯氨酸残基。已知该残基会在分选基序的构象中引入扭结，这对于分选酶结合至关重要。通过引入具有酸或光不稳定骨架修饰的氨基酸，我们计划在SML期间保持扭结构象，然后通过酸处理或UV照射切换到扩展的非活性构象。在最后一个项目中，我们计划联合收割机分选酶和植物转肽酶butelase 1在一个正交的三片段连接方案，使有效的重组表达butelase。考虑到ER定位的酶，我们计划评估和优化butelase在哺乳动物Expi 293细胞和利什曼原虫tarentolae，这两者都已优化ER靶向重组蛋白的表达。应建立乙酰赖氨酸结合模块抑制剂的报告构建体的一锅连接，证明该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.