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PEDS Advance Access originally published online on August 25, 2004
Protein Engineering Design and Selection 2004 17(7):595-602; doi:10.1093/protein/gzh068
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Protein Engineering, Design & Selection vol. 17 no. 7 © Oxford University Press 2004; all rights reserved

A second-generation system for unbiased reading frame selection

Monica L. Gerth1, Wayne M. Patrick1 and Stefan Lutz2

Department of Chemistry and Center for Fundamental and Applied Molecular Evolution, Emory University, 1515 Dickey Drive, Atlanta, GA 30322, USA

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

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.

Received June 23, 2004; revised August 3, 2004; accepted August 13, 2004.

Edited by Andreas Plueckthun


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