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| Natural Extein Free Screening System for Intein Directed-Evolution |
| QIU Pei-ran, HU Fang, LIN Ying, MENG Qing |
| Institute of Biological Sciences and Biotechnology, Donghua University, Shanghai 201620, China |
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Abstract Intein-mediated protein splicing is a brand new solution for protein artificial modification, thus possessing broad application foreground in protein engineering. So far, most inteins present low splicing efficiency in heterogeneous protein which highly limited the intein development and application. In order to develop an high throughput screening system for intein directed-evolution, the Bsa I enzyme was utilized to insert Ter ThyX intein(TX intein) coding sequence into kanamycin resistance protein sequence. Kanamycin screening system can combine the intein-splicing and kanamycin resistance, which is proved by the kanamycin plate assay and Western blot assay. This is an extein free screening system, which is capable of screening the splicing activity and the versatility at the same time. The spliced intein can be screened simply by picking the clone that can survive on the kanamycin plate. The screening system proved to be an efficiency, stable screening system.
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Received: 08 November 2012
Published: 25 January 2013
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