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| Cleavage of Split-Inteins with High Splicing Activity |
| LIN Ying, ZHOU Qian, QUN Qi-jing, MENG Qing |
| Institute of Biological Sciences and Biotechnology,Donghua University,Shanghai 201620,China |
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Abstract Intein-based protein cleavage can be useful tools of recombinant protein purification,ligation and cyclization. However,existing methods were often complicated by low cleavage efficiency,spontaneous cleavages,and low yield of desired protein products. Site-specific cleavages at the N-or C-terminus of an intein can result from alterations of the protein splicing reaction. In N-terminal cleavage,the intein's last residue is mutated to abolish step 3 of the splicing mechanism. Step 1 of the splicing mechanism can still happen,and the resulting ester bond can spontaneously hydrolyze to separate the N-extein from the intein. In C-cleavage,the intein's first residue is mutated to abolish step 1 of the splicing mechanism,but Asn cyclization can still occur to break the peptide bond between the C-extein and intein. The first residue(Cys)or last residue(Asn)mutated split-inteins through site-specific mutagenesis using high splicing S1 and S11 Ssp GyrB split-inteins were constructed and their cleavage efficiency in E.coli cells were tested. Splicing was blocked by site-specific mutation,N-cleavage of S11 split-intein and C-cleavage of S1 split-intein were increased in E.coli cells. This will help us to investigate controllable protein cleavage in vitro,further establish efficient protein purification system and investigate splicing mechanism of split-inteins.
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Received: 13 April 2011
Published: 25 August 2011
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