A second-generation system for unbiased reading frame selection.
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.
Gerth
Monica L
ML
Department of Chemistry and Center for Fundamental and Applied Molecular Evolution, Emory University, 1515 Dickey Drive, Atlanta, GA 30322, USA.
Patrick
Wayne M
WM
Lutz
Stefan
S
D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004331:Drosophila melanogaster; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D005822:Genetic Vectors; D023281:Genomics / Q000379:methods*; D019877:Hydroxymethyl and Formyl Transferases / Q000737:chemistry; D047668:Inteins; D008958:Models, Molecular; D016296:Mutagenesis; D019151:Peptide Library; D051026:Phosphoribosylglycinamide Formyltransferase; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D016364:Reading Frames; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012984:Software
D019151:Peptide Library; D019877:Hydroxymethyl and Formyl Transferases; D051026:Phosphoribosylglycinamide Formyltransferase; D015500:Chloramphenicol O-Acetyltransferase