Skip Navigation



PEDS Advance Access published online on November 2, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh082
This Article
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
17/9/699    most recent
gzh082v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Bertschinger, J.
Right arrow Articles by Neri, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bertschinger, J.
Right arrow Articles by Neri, D.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Received October 4, 2004
Accepted October 23, 2004

Article

Covalent DNA display as a novel tool for directed evolution of proteins in vitro

Julian Bertschinger 1* and Dario Neri 1

1 Institute of Pharmaceutical Sciences, ETH Hönggerberg, Wolfgang-Pauli-Strasse 10, HCI G394, 8093 Zurich, Switzerland

* To whom correspondence should be addressed.
Julian Bertschinger, E-mail: julian.bertschinger{at}pharma.ethz.ch


  Abstract

We present a novel method for the directed evolution of polypeptides, which combines in vitro compartmentalization (Tawfik and Griffiths, 1998) and covalent DNA display. A library of linear DNA fragments is co-packaged with an in vitro transcription/translation mixture in the compartments of a water-in-oil emulsion. Experimental conditions are adjusted so that, in most cases, one compartment contains one DNA molecule. The DNA fragments encode fusion proteins containing a DNA-methyltransferase (M.Hae III), which can form a covalent bond with a 5-fluorodeoxycytosine base at the extremity of the DNA fragment. The resulting library of DNA-protein fusions is extracted from the emulsion, and DNA molecules displaying a protein with desired binding properties are selected from the pool of DNA-protein fusions by affinity panning on target antigens.

We applied this methodology in model selection experiments, using specific ligands for the capture of peptides and globular proteins bound to DNA. We observed enrichment factors >1000-fold for selections performed in separate emulsions, and up to 150-fold for selections performed using mixtures of DNA molecules. M.Hae III could be fused to small globular proteins (such as calmodulin and fibronectin domains), which are ideally suited for the generation of combinatorial libraries and for the isolation of novel binding specificities.

Keywords: DNA display; covalent; compartmentalisation; in vitro; selection.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Protein Eng Des SelHome page
J. Bertschinger, D. Grabulovski, and D. Neri
Selection of single domain binding proteins by covalent DNA display
Protein Eng. Des. Sel., February 1, 2007; 20(2): 57 - 68.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
A. Rothe, R. J. Hosse, and B. E. Power
In vitro display technologies reveal novel biopharmaceutics
FASEB J, August 1, 2006; 20(10): 1599 - 1610.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
J. J. Agresti, B. T. Kelly, A. Jaschke, and A. D. Griffiths
Selection of ribozymes that catalyse multiple-turnover Diels-Alder cycloadditions by using in vitro compartmentalization
PNAS, November 8, 2005; 102(45): 16170 - 16175.
[Abstract] [Full Text] [PDF]


Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.