NDLI: Directed evolution of protein enzymes using nonhomologous random recombination

Content Provider	PubMed Central
Author	Bittker, Joshua A. Le, Brian V. Liu, Jane M. Liu, David R.
Copyright Year	2004
Abstract	We recently reported the development of nonhomologous random recombination (NRR) as a method for nucleic acid diversification and applied NRR to the evolution of DNA aptamers. Here, we describe a modified method, protein NRR, that enables proteins to access diversity previously difficult or impossible to generate. We investigated the structural plasticity of protein folds and the ability of helical motifs to function in different contexts by applying protein NRR and in vivo selection to the evolution of chorismate mutase (CM) enzymes. Functional CM mutants evolved using protein NRR contained many insertions, deletions, and rearrangements. The distribution of these changes was not random but clustered in certain regions of the protein. Topologically rearranged but functional enzymes also emerged from these studies, indicating that multiple connectivities can accommodate a functional CM active site and demonstrating the ability to generate new domain connectivities through protein NRR. Protein NRR was also used to randomly recombine CM and fumarase, an unrelated but also α-helical protein. Whereas the resulting library contained fumarase fragments in many contexts before functional selection, library members surviving selection for CM activity invariably contained a CM core with fumarase sequences found only at the termini or in one loop. These results imply that internal helical fragments cannot be swapped between these proteins without the loss of nearly all CM activity. Our findings suggest that protein NRR will be useful in probing the functional requirements of enzymes and in the creation of new protein topologies.
Related Links	http://dx.doi.org/10.1073/pnas.0402202101
Ending Page	7016
Page Count	6
Starting Page	7011
File Format	PDF
ISSN	00278424
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	18
Volume Number	101
Language	English
Publisher	National Academy of Sciences
Publisher Date	2004-05-04
Access Restriction	Open
Rights Holder	National Academy of Sciences
Subject Keyword	General Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
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4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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