A universal, vector-based system for nucleic acid reading-frame selection.
The identification of a nucleic acid sequence's correct reading frame has important implications for homology-independent protein engineering techniques such as incremental truncation and SCRATCHY. We report the development and experimental implementation of a general in-frame selection system, pSALect, a plasmid vector that utilizes two marker sequences flanking the DNA of interest. This dual selection approach overcomes inconsistencies observed with traditional C-terminally fused reporter proteins. In the pSALect vector, sequences of interest are positioned between an N-terminal Tat-signal sequence and a C-terminal beta-lactamase reporter. In-frame selection of the resulting three-domain protein is performed by growing colonies on ampicillin-containing plates, requiring full-length translation in order to link covalently the signal sequence to the lactamase for export into the periplasm. This dual selection scheme has been validated successfully using defined sequences of glycinamide ribonucleotide formyltransferases (GARTs) from Escherichia coli and human and, in contrast to C-terminal fusion systems, proved effective when applied towards the selection of in-frame constructs in an incremental truncation library.
Lutz
Stefan
S
Department of Chemistry, The Pennsylvania State University, 414 Wartik Laboratory, University Park, PA 16802, USA.
Fast
Walter
W
Benkovic
Stephen J
SJ
D000668:Ampicillin Resistance; D020650:Combinatorial Chemistry Techniques; D004926:Escherichia coli / Q000235:genetics; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D019877:Hydroxymethyl and Formyl Transferases / Q000235:genetics / Q000378:metabolism; D009696:Nucleic Acids; D016366:Open Reading Frames; D019897:Periplasm / Q000378:metabolism; D051026:Phosphoribosylglycinamide Formyltransferase; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
D015696:Gene Products, tat; D009696:Nucleic Acids; D011993:Recombinant Fusion Proteins; D019877:Hydroxymethyl and Formyl Transferases; D051026:Phosphoribosylglycinamide Formyltransferase; D001618:beta-Lactamases