TnTIN and TnTAP: Mini-Transposons for Site-Specific Proteolysis in vivo

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 94; no. 24; pp. 13111 - 13115
• Main Authors: Ehrmann, Michael, Bolek, Petra, Mondigler, Martin, Boyd, Dana, Lange, Roland
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 25.11.1997; National Acad Sciences; National Academy of Sciences; The National Academy of Sciences of the USA
• Subjects: Adenosine Triphosphatases - metabolism; Amino Acid Sequence; Amino acids; Bacterial Proteins - metabolism; Biological Sciences; Codons; DNA Transposable Elements; Endopeptidases - metabolism; Escherichia coli Proteins; Genetic transposition; Genetics; Hydrolysis; Membrane proteins; Membrane Transport Proteins; Molecular Sequence Data; Open reading frames; Plasmids; Proteins; Reading frames; Sandwiches; SEC Translocation Channels; Transposons; Viruses
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.94.24.13111

Tobacco etch virus (TEV) protease recognizes a 7-aa consensus sequence, Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser, where Xaa can be almost any amino acyl residue. Cleavage occurs between the conserved Gln and Ser residues. Because of its distinct specificity, TEV protease can be expressed in the cytoplasm without interfering with viability. Polypeptides that are not natural substrates of TEV protease are proteolyzed if they carry the appropriate cleavage site. Thus, this protease can be used to study target proteins in their natural environment in vivo, as well as in vitro. We describe two Tn5-based mini-transposons that insert TEV protease cleavage sites at random into target proteins. TnTIN introduces TEV cleavage sites into cytoplasmic proteins. TnTAP facilitates the same operation for proteins localized to the bacterial cell envelope. By using two different target proteins, SecA and TolC, we show that such modified proteins can be cleaved in vivo and in vitro by TEV protease. Possible applications of the site-specific proteolysis approach are topological studies of soluble as well as of inner and outer membrane proteins, protein inactivation, insertion mutagenesis experiments, and protein tagging.