Zhao Qing, Zhou Bihong, Gao Xianxing, Xing Lei, Wang Xu, Lin Zhanglin
Department of Chemical Engineering, Tsinghua University, Beijing, China.
Blue Moon Industrial Co. Ltd., Guangzhou, Guangdong, China.
Biotechnol J. 2017 Jun;12(6). doi: 10.1002/biot.201600656. Epub 2017 May 15.
Recombinant protein expression and purification remains a central need for biotechnology. Herein, the authors report a streamlined protein and peptide purification strategy using short self-assembling peptides and a C-terminal cleavage intein. In this strategy, the fusion protein is first expressed as an aggregate induced by the self-assembling peptide. Upon simple separation, the target protein or peptide with an authentic N-terminus is then released in the solution by intein-mediated cleavage. Different combinations of four self-assembling peptides (ELK16, L KD, FK and FR) with three inteins (Sce VMA, Mtu ΔI-CM and Ssp DnaB) were explored. One protein and two peptides were used as model polypeptides to test the strategy. The intein Mtu ΔI-CM, which has pH-shift inducible cleavage, was found to work well with three self-assembling peptides (L KD, FR, FK). Using this intein gave a yield of protein or peptide comparable with that from other more established strategies, such as the Trx-strategy, but in a simpler and more economical way. This strategy provides a simple and efficient method by which to prepare proteins and peptides with an authentic N-terminus, which is especially effective for peptides of 30-100 amino acids in length that are typically unstable and susceptible to degradation in Escherichia coli.
重组蛋白的表达和纯化仍然是生物技术的核心需求。在此，作者报告了一种使用短自组装肽和C端切割内含肽的简化蛋白质和肽纯化策略。在该策略中，融合蛋白首先作为由自组装肽诱导形成的聚集体表达。经过简单分离后，具有天然N端的目标蛋白或肽通过内含肽介导的切割在溶液中释放出来。研究了四种自组装肽（ELK16、L KD、FK和FR）与三种内含肽（Sce VMA、Mtu ΔI-CM和Ssp DnaB）的不同组合。使用一种蛋白质和两种肽作为模型多肽来测试该策略。发现具有pH值变化诱导切割功能的内含肽Mtu ΔI-CM与三种自组装肽（L KD、FR、FK）配合良好。使用这种内含肽获得的蛋白质或肽产量与其他更成熟的策略（如Trx策略）相当，但方式更简单、更经济。该策略提供了一种简单有效的方法来制备具有天然N端的蛋白质和肽，这对于长度为30 - 100个氨基酸的肽尤其有效，这些肽通常不稳定且在大肠杆菌中易降解。
