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General Method for Sequence-independent Site-directed Chimeragenesis

Hiraga, Kaori and Arnold, Frances H. (2003) General Method for Sequence-independent Site-directed Chimeragenesis. Journal of Molecular Biology, 330 (2). pp. 287-296. ISSN 0022-2836. doi:10.1016/S0022-2836(03)00590-4.

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We have developed a simple and general method that allows for the facile recombination of distantly related (or unrelated) proteins at multiple discrete sites. To evaluate the sequence-independent site-directed chimeragenesis (SISDC) method, we have recombined β-lactamases TEM-1 and PSE-4 at seven sites, examined the quality of the chimeric genescreated, and screened the library of 2^8 (256) chimeras for functional enzymes. Probe hybridization and sequencing analyses revealed that SISDC generated a random library with little sequence bias and in which all targeted fragments were recombined in the desired order. Sequencing the genes from clones having functional lactamases identified 14 unique chimeras. These chimeras are characterized by a lower level of disruption, as calculated by the SCHEMA algorithm, than the library as a whole. These results illustrate the use of SISDC in creating designed chimeric protein libraries and further illustrate the ability of SCHEMA to identify chimeras whose folded structures are likely not to be disrupted by recombination.

Item Type:Article
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Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2003 Elsevier Science Ltd. Received 11 February 2003, Revised 27 April 2003, Accepted 28 April 2003, Available online 25 June 2003. We thank Christopher A. Voigt for the SCHEMA calculation, and Jeffrey Endelman and Jonathan Silberg for helpful discussions on data analysis. This research was supported, in part, by a JSPS Postdoctoral Fellowship for Research Abroad (to K.H.).
Funding AgencyGrant Number
Japan Society for the Promotion of Science (JSPS)UNSPECIFIED
Subject Keywords:DNA shuffling; non-homologous recombination; directed evolution; SCHEMA
Issue or Number:2
Record Number:CaltechAUTHORS:20180822-085748088
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Official Citation:Kaori Hiraga, Frances H. Arnold, General Method for Sequence-independent Site-directed Chimeragenesis, Journal of Molecular Biology, Volume 330, Issue 2, 2003, Pages 287-296, ISSN 0022-2836, (
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:89012
Deposited By: Tony Diaz
Deposited On:22 Aug 2018 16:35
Last Modified:16 Nov 2021 00:31

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