Novel split intein for trans-splicing synthetic peptide onto C terminus of protein.
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 (IN) and a 6-aa C-intein (IC). 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)x10(-5) s(-1) and reached a high efficiency of approximately 80%. This S11 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.
Appleby
Julia H
JH
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada.
Zhou
Kaisong
K
Volkmann
Gerrit
G
Liu
Xiang-Qin
XQ
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D027081:DNA Gyrase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D047668:Inteins / Q000502:physiology*; D015202:Protein Engineering / Q000379:methods; D019154:Protein Splicing / Q000502:physiology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D046939:Synechocystis / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D027081:DNA Gyrase