从药物多肽到蛋白质全合成：酶促拼接的方法原理与前沿应用

杨新宇; 朱彤; 李瑞峰; 吴边

doi:10.12211/2096-8280.2020-064

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从药物多肽到蛋白质全合成：酶促拼接的方法原理与前沿应用

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- 从药物多肽到蛋白质全合成：酶促拼接的方法原理与前沿应用
- Enzymatic ligation technologies for the synthesis of pharmaceutical peptides and proteins
- 合成生物学 2021年2卷第1期页码：33-45
- 作者机构：
  
  1.中国科学院微生物研究所，中国科学院微生物生理与代谢工程重点实验室，微生物资源前期开发国家重点实验室，北京 100101
  2.中国科学院大学生命科学学院，北京 100049
- 作者简介：
  
  [ "杨新宇（1994—），男，硕士研究生，研究方向为酶促蛋白质化学合成与修饰。E-mail：yangxy@im.ac.cn" ]
  [ "吴边（1982—），男，博士生导师，研究员，研究方向为微生物催化元件的深度挖掘、机理解析、合成设计。致力于将蛋白质计算机设计前沿方法引入酶工程的研究，改造复杂生物大分子结构，优化催化元件性能，并在此基础上构建重要药物前体的生物合成途径。E-mail：wub@im.ac.cn" ]
- 基金信息：
  
  国家重点研发计划(2018YFA0901600);国家自然科学基金优秀青年科学基金(31822002);国家自然科学基金面上项目(31870055)
- DOI：10.12211/2096-8280.2020-064
  中图分类号： Q51
- 收稿：2020-05-18，
  
  修回：2021-01-06，
  
  纸质出版：2021-02-28
- 稿件说明：
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杨新宇, 朱彤, 李瑞峰, 吴边. 从药物多肽到蛋白质全合成：酶促拼接的方法原理与前沿应用[J]. 合成生物学, 2021, 2(1): 33-45

YANG Xinyu, ZHU Tong, LI Ruifeng, WU Bian. Enzymatic ligation technologies for the synthesis of pharmaceutical peptides and proteins[J]. Synthetic Biology Journal, 2021, 2(1): 33-45
杨新宇, 朱彤, 李瑞峰, 吴边. 从药物多肽到蛋白质全合成：酶促拼接的方法原理与前沿应用[J]. 合成生物学, 2021, 2(1): 33-45 DOI： 10.12211/2096-8280.2020-064.

YANG Xinyu, ZHU Tong, LI Ruifeng, WU Bian. Enzymatic ligation technologies for the synthesis of pharmaceutical peptides and proteins[J]. Synthetic Biology Journal, 2021, 2(1): 33-45 DOI： 10.12211/2096-8280.2020-064.

摘要

蛋白质是生命活动的基础功能元件，其化学合成与定点修饰已成为合成生物学领域探索复杂生物大分子“结构-功能”关系的重要前沿方向。近年来，以多肽固相合成与特异性拼接为核心的蛋白质合成和修饰技术蓬勃发展，打破了生命合成系统仅能使用天然及少数非天然氨基酸的瓶颈，为制备含有数百个氨基酸残基的非天然蛋白质提供了技术平台，让原子水平的蛋白质人工设计成为现实。作为一类广受关注的多肽拼接策略，基于天然或人工改造多肽连接酶的技术方法不仅在基础研究领域拓展了人们对蛋白质这一生命核心元件的理解，还在工业领域崭露头角，被应用于多种多肽类药物的生产。针对蛋白质合成领域中酶促多肽拼接技术平台，本文介绍了Sortase A转肽酶、Butelase 1转肽酶以及Subtilisin人工连接酶的来源以及催化过程，探讨了各自的优势以及局限性，并综述了三种酶在蛋白质修饰、蛋白质合成、多肽药物环化等方面的应用。通过计算机辅助设计、定向进化等技术对转肽酶、连接酶进行改造来提升其在底物谱、催化活性等方面的特性，将化学方法与酶促方法联用来建立多样的生物大分子从头设计与合成路线是目前的主要发展趋势。

Abstract

As a hot-spot of synthetic biology

chemical protein synthesis and modification have been widely applied to generate and functionalize naturally inaccessible proteins to meet scientific or pharmaceutical demands. Breaking away from the restrictions on the amino acids utilized in ribosomal protein synthesis

protein synthesis

and modification through the ligation of synthetic peptides have provided a platform for preparing the unnatural proteins that contain hundreds of residues

which realizes the artificial design of protein on the atomic scale. Although several chemical ligation methods have been reported

they are confronted with the defects like limited junction choices and complicated substrate preparations

impelling the scientists to find out alternative solutions and develop enzymatic strategies fit for different applications. As a group of popular peptide ligation methods

enzymatic ligation strategi

es not only expand our understanding of protein

but also exhibit great advantages in the industrial production of pharmaceutical peptides.Sortase A (SrtA) has been extensively utilized for protein terminal modification. The researchers from various fields have attempted to develop novel applications based on SrtA

for instance

characterizing the surface proteins of eukaryotic cells and expanding the choices of phage capsid proteins. Butelase 1 from a cyclopeptide-producing plant (

Clitoria ternatea

) is a highly efficient peptide ligase for peptide cyclization. Large proteins like GFP could be cyclized with excellent yield (

95%) as well

and Butelase 1 can be used for synthesizing peptide dendrimers

preparing protein thioesters

and modifying bacterial surfaces as well. Subtilisin-derived ligase is engineered from subtilisin and thought as a powerful tool for peptide cyclization

protein synthesis and terminal modification. In recent years

based on a calcium-independent subtilisin variant

a thermostable and organic solvent-tolerant peptide ligase was constructed and termed Peptiligase

which was further engineered afterward. Omniligase-1 is a universal ligase bearing broad sequence compatibility

while Thymoligase is specially designed for the production of the pharmaceutic peptide Thymosin-α1. These Peptiligase variants exhibit great advantages for industrial applications and have been considered as the most impressive enzymatic approach to date. The development of the existing enzymatic methods is anticipated to be ameliorated through enzyme engineering

to which rational design and directed evolution have contributed a lot. At the moment of computational protein design communicating with chemical protein synthesis

we anticipate that more peptide ligases would be discovered or designed. They may serve for generations of previously hard-to-access modified proteins.

关键词

Keywords

references

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