Skip Navigation

This Article
Right arrow Full Text Freely available
Right arrow Print PDF (702K) Freely available
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
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Lutz, S.
Right arrow Articles by Benkovic, S. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lutz, S.
Right arrow Articles by Benkovic, S. J.
Related Collections
Right arrow Mutagenesis
Right arrow Recombinant DNA expression
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research, 2001, Vol. 29, No. 4 e16
© 2001 Oxford University Press

Rapid generation of incremental truncation libraries for protein engineering using {alpha}-phosphothioate nucleotides

Stefan Lutz, Marc Ostermeier and Stephen J. Benkovic*

Department of Chemistry, The Pennsylvania State University, 414 Wartik Laboratory, University Park, PA 16802, USA

Incremental truncation for the creation of hybrid enzymes (ITCHY) is a novel tool for the generation of combinatorial libraries of hybrid proteins independent of DNA sequence homology. We herein report a fundamentally different methodology for creating incremental truncation libraries using nucleotide triphosphate analogs. Central to the method is the polymerase catalyzed, low frequency, random incorporation of {alpha}-phosphothioate dNTPs into the region of DNA targeted for truncation. The resulting phosphothioate internucleotide linkages are resistant to 3'->5' exonuclease hydrolysis, rendering the target DNA resistant to degradation in a subsequent exonuclease III treatment. From an experimental perspective the protocol reported here to create incremental truncation libraries is simpler and less time consuming than previous approaches by combining the two gene fragments in a single vector and eliminating additional purification steps. As proof of principle, an incremental truncation library of fusions between the N-terminal fragment of Escherichia coli glycinamide ribonucleotide formyltransferase (PurN) and the C-terminal fragment of human glycinamide ribonucleotide formyltransferase (hGART) was prepared and successfully tested for functional hybrids in an auxotrophic E.coli host strain. Multiple active hybrid enzymes were identified, including ones fused in regions of low sequence homology.

* To whom correspondence should be addressed. Tel: +1 814 865 2882; Fax: +1 814 865 2973; Email: sjb1{at}psu.edu Present address: Marc Ostermeier, Department of Chemical Engineering, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218-2694, USA


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
 Nucleic Acids ResHome page
Z. Yang, A. M. Sismour, and S. A. Benner
Nucleoside alpha-thiotriphosphates, polymerases and the exonuclease III analysis of oligonucleotides containing phosphorothioate linkages
Nucleic Acids Res., May 14, 2007; 35(9): 3118 - 3127.
[Abstract] [Full Text] [PDF]

Home page
 Appl. Environ. Microbiol.Home page
K. Mori, T. Mukaihara, Y. Uesugi, M. Iwabuchi, and T. Hatanaka
Repeat-Length-Independent Broad-Spectrum Shuffling, a Novel Method of Generating a Random Chimera Library In Vivo
Appl. Envir. Microbiol., February 1, 2005; 71(2): 754 - 760.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. W. Deem
Evolution and evolvability of proteins in the laboratory
PNAS, March 23, 2004; 101(12): 3997 - 3998.
[Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. C. Saraf, A. R. Horswill, S. J. Benkovic, and C. D. Maranas
From the Cover: FamClash: A method for ranking the activity of engineered enzymes
PNAS, March 23, 2004; 101(12): 4142 - 4147.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
C. Neylon
Chemical and biochemical strategies for the randomization of protein encoding DNA sequences: library construction methods for directed evolution
Nucleic Acids Res., February 27, 2004; 32(4): 1448 - 1459.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
Y. Kawarasaki, K. E. Griswold, J. D. Stevenson, T. Selzer, S. J. Benkovic, B. L. Iverson, and G. Georgiou
Enhanced crossover SCRATCHY: construction and high-throughput screening of a combinatorial library containing multiple non-homologous crossovers
Nucleic Acids Res., November 1, 2003; 31(21): e126 - e126.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
G. L. Moore and C. D. Maranas
Identifying residue-residue clashes in protein hybrids by using a second-order mean-field approach
PNAS, April 29, 2003; 100(9): 5091 - 5096.
[Abstract] [Full Text] [PDF]

Home page
 Protein Eng Des SelHome page
S. Lutz, W. Fast, and S. J. Benkovic
A universal, vector-based system for nucleic acid reading-frame selection
Protein Eng. Des. Sel., December 1, 2002; 15(12): 1025 - 1030.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
T. A. Naumann, I. Y. Goryshin, and W. S. Reznikoff
Production of combinatorial libraries of fused genes by sequential transposition reactions
Nucleic Acids Res., November 1, 2002; 30(21): e119 - e119.
[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.