Open AccessFacile approach for constructing TEV insertions to probe protein structure in vivo
Author(s) -
Elysa Brown,
Stanley Maloy
Publication year - 2006
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/000112284
Subject(s) - biology , terminator (solar) , transposable element , gene , genetics , open reading frame , operon , transposon mutagenesis , reporter gene , plasmid , lac operon , computational biology , gene expression , peptide sequence , escherichia coli , genome , ionosphere , physics , astronomy
The tobacco etch virus (TEV) protease has been used as a tool to examine protein structure in vivo. TEV cleavage sites (TEVcs) have been introduced via cloning into unique restriction sites or random transposon mutagenesis. We describe a facile, efficient method for introducing TEVcs at precise locations in a gene to test specific predictions about protein structure. The method uses the λ Red recombination system to construct seamless, in-frame insertions of the TEVcs at any desired location within an open reading frame (ORF). The system was tested using the multifunctional PutA protein Salmonella enterica sv. Typhimurium. The first step involved insertion of a chloramphenicol resistance (Cam R ) cassette with a transcriptional terminator at the desired location. A second swap then replaces the Cam R insertion with the TEVcs. Placing a copy of the lac operon downstream of the putA gene provides a simple counterselection for replacement of the Cam R insertion and also provides a reporter gene for monitoring transcription of the mutated gene.