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| High Splicing Activity of Ter DnaE-3 Mini-intein through Directed Evolution |
| XUN Qi-jing, LIN Ying, QIU Pei-ran, MENG Qing |
| Institute of Biological Sciences and Biotechnology, Donghua University, Shanghai 201620, China |
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Abstract Natural and artificially engineered inteins have been more widely used in the field of protein engineering. However, inteins are often inactive when cloned into a heterologous host protein, and also need the presence of the native exteins, which would be left in the target protein. Inteins should overcome these limitations. In order to improve splicing activity of Ter DnaE-3 (Trichodesmium erythraeum) mini-intein in heterologous host, random mutagenesis was based on error PCR by change the concentration of dNTP、Mg2+、Mn2+, and then mutants were screened in kanamycin resistance-dependent system. After directed evolution, mutant 5th increased splicing activity from ~20% to ~85%, and mutant 9th can avoid occurring cleavage compared with other mutants by western blot analysis. The relationship of amino acid mutations and splicing activity showed that amino acids involved in the formation of α-helix structure may influence intein cleavage reaction, and amino acids involved in the formation of β-sheet structure may provide some help for intein structure. The resulting improved inteins showed high activity in heterologous host, which verify the kanamycin resistance-dependent system feasibility and expand intein application.
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Received: 30 December 2011
Published: 25 May 2012
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