期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 284, 期 10, 页码 6194-6199出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M805474200
关键词
-
资金
- Canadian Institutes of Health Research
Conventional split inteins have been useful for trans-splicing between recombinant proteins, and an artificial S1 split intein is useful for adding synthetic peptide onto the N terminus of recombinant proteins. Here we have engineered a novel S11 split intein for trans-splicing synthetic peptide onto the C terminus of recombinant proteins. The C-intein of the S11 split intein is extremely small (6 amino acids (aa)); thus it can easily be produced together with a synthetic C-extein to be added to the C terminus of target proteins. The S11 intein was derived from the Ssp GyrB intein after deleting the homing endonuclease domain and splitting the remaining intein sequence near the C terminus, producing a 150-aa N-intein (I-N) and a 6-aa C-intein (I-C). Its trans-splicing activity was demonstrated first in Escherichia coli cells and then in vitro for trans-splicing between a synthetic peptide and a recombinant protein. The in vitro trans-splicing reaction exhibited a typical rate constant of (6.9 +/- 2.2) x 10(-5) s(-1) and reached a high efficiency of similar to 80%. This S-1 split intein can be useful for adding any desirable chemical groups to the C terminus of a protein of interest, which may include modified and unnatural amino acids, biotin and fluorescent labels, and even drug molecules.
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