Engineered pH-inducible intein Mtu ΔI-CM variants with markedly reduced premature cleavage activity

作者：Lin, Zhanglin^*; Zhao, Qing; Wang, Xu; Zhou, Bihong; Xing, Lei; Wang, Jiangyun; Pistolozzi, Marco; Zhao, Lei; Wang, Tingting^*

来源：AICHE Journal, 2020, 66(3): e16806.

DOI：10.1002/aic.16806

• C-terminal cleavage
• directed evolution
• intein
• protein engineering
• tagless protein

Inteins have been widely exploited for the purification of tagless proteins. Among them, pH-inducible C-terminal-cleavage inteins enable the preparation of proteins and peptides with an authentic N-terminus. However, a severe premature cleavage around neutral pH has limited the application of these inteins, especially when used in recombinant hosts such as Escherichia coli. By targeting the microenvironment of the two key histidine residues H429 and H439, we engineered Mtu Delta I-CM intein to markedly reduce its premature cleavage. Kinetic analyses suggested that although the variants retained the pH dependence, they indeed cleaved slower, especially at pH 7.6. These variants resulted in higher yields for two model polypeptides than the original Mtu Delta I-CM intein, when used in conjunction with a cleavable self-assembling tag. This work suggests that more controllable pH-inducible inteins can be obtained by manipulating the residues in the self-cleavage sites and provide better performance for tag-based protein preparation strategies.

• 单位
  中国科学院生物物理研究所; 清华大学