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Evolving Proteins of Novel Composition

Montclare, Jin Kim and Tirrell, David A. (2006) Evolving Proteins of Novel Composition. Angewandte Chemie International Edition, 45 (27). pp. 4518-4521. ISSN 1433-7851. doi:10.1002/ange.200600088.

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The behavior of proteins can be altered significantly by the incorporation of noncanonical amino acids. Changes in spectroscopic properties, thermal stability, and molecular recognition behavior have been reported. In many cases, however, introduction of novel amino acids causes loss of protein function. Herein we describe a new approach to protein engineering in which amino acid replacement is combined with directed evolution to create functional proteins of novel composition. Global replacement of the leucine residues of chloramphenicol acetyltransferase (CAT) by 5’,5’,5’-trifluoroleucine (TFL) results in a 20-fold reduction in the half-life (t_(1/2)) of thermal inactivation of the enzyme at 608C. Two rounds of random mutagenesis and screening yielded a variant of CAT containing three amino acid substitutions, which in fluorinated form demonstrates a 27-fold improvement in t_(1/2), recovering the loss in thermostability caused by fluorination.

Item Type:Article
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URLURL TypeDescription Information
Tirrell, David A.0000-0003-3175-4596
Additional Information:© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Received: January 9, 2006. Published online: June 8, 2006. We thank Frances Arnold for use of several instruments. This work was supported by NIH GM 62523 and 5F32 GM67375-2 (D.A.T. and J.K.M.) and by the Beckman Institute of the California Institute of Technology(D.A.T .).
Funding AgencyGrant Number
NIH Postdoctoral Fellowship5F32 GM67375-2
Caltech Beckman InstituteUNSPECIFIED
Subject Keywords:amino acids · directed evolution · enzymes · protein engineering · thermostability
Issue or Number:27
Record Number:CaltechAUTHORS:MONacie2006
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Official Citation:Montclare, J. K. and Tirrell, D. A. (2006), Evolving Proteins of Novel Composition. Angew. Chem., 118: 4630–4633. doi: 10.1002/ange.200600088
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:53246
Deposited By: Anne Hormann
Deposited On:16 Jan 2015 04:43
Last Modified:02 Nov 2021 18:12

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