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Engineered pH‐inducible intein Mtu ΔI‐CM variants with markedly reduced premature cleavage activity
Author(s) -
Lin Zhanglin,
Zhao Qing,
Wang Xu,
Zhou Bihong,
Xing Lei,
Wang Jiangyun,
Pistolozzi Marco,
Zhao Lei,
Wang Tingting
Publication year - 2020
Publication title -
aiche journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.958
H-Index - 167
eISSN - 1547-5905
pISSN - 0001-1541
DOI - 10.1002/aic.16806
Subject(s) - intein , cleavage (geology) , recombinant dna , chemistry , escherichia coli , histidine , biochemistry , combinatorial chemistry , biophysics , amino acid , biology , gene , paleontology , rna , fracture (geology) , rna splicing
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 ΔI‐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 ΔI‐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.