Appleby Julia H, Zhou Kaisong, Volkmann Gerrit, Liu Xiang-Qin
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada.
J Biol Chem. 2009 Mar 6;284(10):6194-9. doi: 10.1074/jbc.M805474200. Epub 2009 Jan 9.
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.
传统的分裂内含肽已被用于重组蛋白之间的反式剪接，而人工合成的S1分裂内含肽则可用于在重组蛋白的N端添加合成肽。在此，我们设计了一种新型的S11分裂内含肽，用于在重组蛋白的C端反式剪接合成肽。S11分裂内含肽的C-内含肽极小（6个氨基酸），因此它能很容易地与合成的C-外显肽一起产生，以便添加到靶蛋白的C端。S11内含肽是在删除归巢内切酶结构域并在C端附近分裂剩余的内含肽序列后，从Ssp GyrB内含肽衍生而来，产生了一个150个氨基酸的N-内含肽（IN）和一个6个氨基酸的C-内含肽（IC）。其反式剪接活性首先在大肠杆菌细胞中得到证明，然后在体外证明了其在合成肽与重组蛋白之间的反式剪接。体外反式剪接反应的典型速率常数为(6.9±2.2)×10(-5) s(-1)，效率高达约80%。这种S11分裂内含肽可用于向目标蛋白的C端添加任何所需的化学基团，这些基团可能包括修饰的和非天然的氨基酸基团、生物素和荧光标签，甚至药物分子。
