Romero, Philip A. and Stone, Everett and Lamb, Candice and Chantranupong, Lynne and Krause, Andreas and Miklos, Alexandr E. and Hughes, Randall A. and Fechtel, Blake and Ellington, Andrew D. and Arnold, Frances H. and Georgiou, George (2012) SCHEMA-Designed Variants of Human Arginase I and II Reveal Sequence Elements Important to Stability and Catalysis. ACS Synthetic Biology, 1 (6). pp. 221-228. ISSN 2161-5063 http://resolver.caltech.edu/CaltechAUTHORS:20120716-102139957
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Arginases catalyze the divalent cation-dependent hydrolysis of l-arginine to urea and l-ornithine. There is significant interest in using arginase as a therapeutic anti-neogenic agent against l-arginine auxotrophic tumors and in enzyme replacement therapy for treating hyperargininemia. Both therapeutic applications require enzymes with sufficient stability under physiological conditions. To explore sequence elements that contribute to arginase stability we used SCHEMA-guided recombination to design a library of chimeric enzymes composed of sequence fragments from the two human isozymes Arginase I and II. We then developed a novel active learning algorithm that selects sequences from this library that are both highly informative and functional. Using high-throughput gene synthesis and our two-step active learning algorithm, we were able to rapidly create a small but highly informative set of seven enzymatically active chimeras that had an average variant distance of 40 mutations from the closest parent arginase. Within this set of sequences, linear regression was used to identify the sequence elements that contribute to the long-term stability of human arginase under physiological conditions. This approach revealed a striking correlation between the isoelectric point and the long-term stability of the enzyme to deactivation under physiological conditions.
|Additional Information:||© 2012 American Chemical Society. Published In Issue June 15, 2012; Article ASAP April 10, 2012; Just Accepted Manuscript March 30, 2012; Received: March 07, 2012. This project was supported by grants (#HF0032 and F-1654) from TI3D/Welch Foundation and National Institutes of Health (CA 139059). In addition, this work was supported by the National Security Science and Engineering Faculty Fellowship (FA9550-10-1-0169), and L.C. was supported by a fellowship from the Arnold & Mabel Beckman Foundation. The authors also acknowledge the National Institutes of Health, ARRA (grant R01-GM068664 to FHA) for funding SCHEMA library design, and the U.S. Army Research Office, Institute for Collaborative Biotechnologies (grant W911NF-09-D-0001 to FHA) for funding the regression analysis work. These contents are solely the responsibility of the authors and do not necessarily represent the official views of the sponsors.|
|Subject Keywords:||enzyme engineering; arginase; homologous recombination; SCHEMA library design; active learning; protein stability|
|Official Citation:||SCHEMA-Designed Variants of Human Arginase I and II Reveal Sequence Elements Important to Stability and Catalysis Philip A. Romero, Everett Stone, Candice Lamb, Lynne Chantranupong, Andreas Krause, Aleksandr E. Miklos, Randall A. Hughes, Blake Fechtel, Andrew D. Ellington, Frances H. Arnold, and George Georgiou ACS Synthetic Biology 2012 1 (6), 221-228|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Jason Perez|
|Deposited On:||16 Jul 2012 17:56|
|Last Modified:||26 Dec 2012 15:33|
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