Synthetic biology engineering to catalyse unbreakable linkage between peptide building blocks
Synthetic biology engineering to catalyse unbreakable linkage between peptide building blocks
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合成生物工程催化肽结构单元之间牢不可破的连接
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发表时间：
2017
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通讯作者：
Can M Buldun
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作者：
Can M Buldun
蛋白质偶联是研究和开发新疗法的重要工具。肽标签是生物缀合的理想手柄，因为它们对蛋白质结构和功能的破坏性最小，并且免疫原性弱。在这篇论文中，我开发了SnoopLigase，一种能够通过位点特异性转酰胺作用共价连接两个肽标签SnoopTagJr和DogTag的蛋白质。这三个模块是通过裂解肺炎链球菌粘附素RrgA的含异肽键结构域获得的。使用合理的和计算的蛋白质设计，提高了反应的速率和产率，同时允许在不同的缓冲液和温度下进行有效的反应。当使用已建立的生物缀合技术时，缀合的蛋白质与未反应的底物的分离可能具有挑战性。SnoopLigase与其反应产物强烈结合，这使得能够通过SnoopLigase固相固定和随后的产物洗脱来纯化缀合的SnoopTagJr-DogTag。因此，SnoopLigase可用于生产高质量的生物缀合物治疗剂。疫苗是重要的医疗干预措施，每年可挽救数百万人的生命。人工生产的抗原具有成本效益，并且具有无与伦比的安全性，但通常显示出较差的免疫原性。蛋白质多聚化可以强烈增强针对抗原的免疫应答。基于SnoopLigase系统和七聚体蛋白IMX 313，我开发了一个高度稳定的模块化疫苗平台。SnoopTagJr连接的蛋白质与平台纳米颗粒顶X 313-DogTag的共价缀合允许简单、有效和模块化的蛋白质多聚化。SnoopLigase的有效偶联和小尺寸的标签提供了加速开发针对疟疾和其他疾病的模块化疫苗的潜力。
Protein conjugation is an important tool for research and the development of novel therapeutics. Peptide tags are ideal handles for bioconjugation, as they are minimally disruptive to protein structure and function and are weakly immunogenic. In this thesis, I have developed SnoopLigase, a protein which is able to covalently link the two peptide tags SnoopTagJr and DogTag by site-specific transamidation. The three modules were obtained by splitting an isopeptide bond-containing domain of the Streptococcus pneumoniae adhesin RrgA. Using rational and computational protein design, rate and yield of the reaction were improved, while allowing efficient reaction in different buffers and temperatures. The separation of conjugated proteins from unreacted substrate can be challenging when using established bioconjugation techniques. SnoopLigase bound strongly to its reaction product, which enabled the purification of conjugated SnoopTagJr-DogTag by SnoopLigase solid-phase immobilisation and subsequent product elution. Hence, SnoopLigase could be useful to produce high quality bioconjugate therapeutics. Vaccines are important medical interventions, saving millions of lives each year. Recombinantly produced antigens are cost-effective and have an unmatched safety profile, yet often show poor immunogenicity. Protein multimerisation can strongly enhance the immune response against the antigen. Based on the SnoopLigase system and the heptameric protein IMX313, I developed a highly stable, modular vaccine platform. Covalent conjugation of SnoopTagJr-linked proteins to the platform nanoparticle IMX313-DogTag allowed simple, efficient and modular protein multimerisation. The efficient coupling by SnoopLigase and the small size of the tags provide potential to accelerate development of modular vaccines against malaria and other diseases.