title: An intein-cassette integration approach used for the generation of a split TEV protease activated by conditional protein splicing.
abstract: Ligand-induced conditional protein splicing (CPS) using a split intein allows the covalent reconstitution of a protein from two polypeptide fragments. The small molecule rapamycin binds to the fused FKBP and FRB dimerizer domains and thereby induces folding of the split intein, which then removes itself in the trans-splicing reaction. CPS has great potential for the experimental control of protein activity in living cells, however, only one such example was reported yet. This discrepancy is due to the challenging reconstitution of a protein from two inactive fragments because of folding, stability, and solubility issues. Moreover, in CPS the split intein must be active in the specific sequence context. We here report the novel concept, design, and application of a CPS cassette for facile target gene modification to identify active split intein insertion sites. The CPS cassette encodes the split intein and dimerizer domain gene fragments as well as a selectable genetic marker for yeast. The addition of short sequences in the PCR-amplification of the CPS cassette allowed its site-specific insertion into the target gene by homologous recombination. Our approach thus avoids the extensive DNA cloning steps typically required. By this strategy, we identified two CPS variants of the tobacco etch virus (TEV) protease that are conditionally activated by rapamycin in yeast and we show their potential for the manipulation of intracellular proteins through proteolysis events. Our results suggest that more proteins will be amenable to CPS control and that intein cassette integration is a powerful tool for the development of such conditional variants as well as for other application of cis- and trans-splicing inteins.
authors.affiliation: TU Dortmund University, Faculty of Chemistry-Chemical Biology, Otto-Hahn-Str. 6, 44227 Dortmund, Germany.
authors.forename: Tim
authors.initials: T
authors.lastname: Sonntag
authors.forename: Henning D
authors.initials: HD
authors.lastname: Mootz
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D047668:Inteins / Q000235:genetics*; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering; D019154:Protein Splicing; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D059010:Single-Cell Analysis; D020123:Sirolimus / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D010450:Endopeptidases; C099488:TEV protease; D020123:Sirolimus