TNTIN AND TNTAP - MINI-TRANSPOSONS FOR SITE-SPECIFIC PROTEOLYSIS IN-VIVO

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 Gin and Ser residues, B ecause of its distinct specificity, TEV protease can be expressed in t he 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 stud y target proteins in their natural environment in vivo, as well as in vitro. We describe two Tn5-based mini-transposons that insert TEV prot ease cleavage sites at random into target proteins, TnTIN introduces T EV cleavage sites into cytoplasmic proteins, TnTAP facilitates the sam e 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 t opological studies of soluble as well as of inner and outer membrane p roteins, protein inactivation, insertion mutagenesis experiments, and protein tagging.