This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Song, J. K. Articles by Rhee, J. S. Search for Related Content PubMed PubMed Citation Articles by Song, J. K. Articles by Rhee, J. S. Agricola Articles by Song, J. K. Articles by Rhee, J. S.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, December 2002, p. 6146-6151, Vol. 68, No. 12
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.12.6146-6151.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase

Jae Kwang Song, Bora Chung, Young Hak Oh, and Joon Shick Rhee^*

Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejon 305-701, Korea

Received 14 June 2002/ Accepted 25 September 2002

A method of mutagenic and unidirectional reassembly (MURA) that can generate libraries of DNA-shuffled and randomly truncated proteins was developed. The method involved fragmenting the template gene(s) randomly by DNase I and reassembling the small fragments with a unidirectional primer by PCR. The MURA products were treated with T4 DNA polymerase and subsequently with a restriction enzyme whose site was located on the region of the MURA primer. The N-terminal-truncated and DNA-shuffled library of a Serratia sp. phospholipase A₁ prepared by this method had an essentially random variation of truncated size and also showed point mutations associated with DNA shuffling. After high-throughput screening on triglyceride-emulsified plates, several mutants exhibiting absolute lipase activity (NPL variants) were obtained. The sequence analysis and the lipase activity assay on the NPL variants revealed that N-terminal truncations at a region beginning with amino acids 61 to 71, together with amino acid substitutions, resulted in the change of substrate specificity from a phospholipase to a lipase. We therefore suggest that the MURA method, which combines incremental truncation with DNA shuffling, can contribute to expanding the searchable sequence space in directed evolution experiments.

---

* Corresponding author. Mailing address: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejon 305-701, Korea. Phone: 82-42-869-2613. Fax: 82-42-869-2610. E-mail: jsrhee{at}mail.kaist.ac.kr.

Present address: Applied and Engineering Chemistry Division, Korea Research Institute of Chemical Technology, Yuseong-gu, Daejon 305-600, Korea.

---

Applied and Environmental Microbiology, December 2002, p. 6146-6151, Vol. 68, No. 12
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.12.6146-6151.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Yuan, L., Kurek, I., English, J., Keenan, R. (2005). Laboratory-Directed Protein Evolution. Microbiol. Mol. Biol. Rev. 69: 373-392 [Abstract] [Full Text]  
• Osuna, J., Yanez, J., Soberon, X., Gaytan, P. (2004). Protein evolution by codon-based random deletions. Nucleic Acids Res 32: e136-e136 [Abstract] [Full Text]