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PEDS Advance Access published online on August 25, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh068
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Received June 23, 2004
Revised August 3, 2004
Accepted August 13, 2004

Article

"A second generation system for unbiased reading frame selection"

Monica L. Gerth , Wayne M. Patrick , Stefan Lutz *

* To whom correspondence should be addressed. E-mail: sal2{at}emory.edu.


  Abstract

Reading frame selection of nucleic acids has important implications for protein engineering and genomics. Current methods are limited because selection of the gene of interest inevitably depends on the solubility of its translated product. Here we report the construction of the pInSALect vector, which provides strict reading frame selection without concomitant selection for protein solubility or folding. This plasmid incorporates the cis-splicing VMA intein sequence from Saccharomyces cerevisiae to facilitate the post-translational self-excision of the protein of interest, thereby eliminating potential aggregation problems. Results from two libraries of chimeric glycinamide ribonucleotide formyltransferases confirm the superior performance of pInSALect over existing reading frame selection systems.

Keywords: protein engineering; directed evolution; methodology; selection; intein.
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