Controlling splicing and cleavage led to understanding protein splicing. Several chapters of this volume will describe applications based on protein splicing. These applications are predicated on the ability to turn inteins on and off by mutation, temperature-dependent or pH-sensitive splicing, placing inteins in suboptimal insertion sites, chemically caging nucleophiles, thiols, zinc, splitting inteins, or activating split inteins with small molecules.
Most applications involve using inteins to purify proteins and for protein semisynthesis. Protein purification is usually accomplished by mutating the intein so that it will cleave on demand the splice junction between the intein and the protein of interest. A purification tag or a folding reporter is often added to one end of the intein or in place of the homing endonu-clease domain. Protein semisynthesis can be performed directly with inteins using trans-splicing or it can be indirectly performed by taking advantage of the carboxy-terminal a-thioester produced by thiol cleavage of a target protein linked to the intein amino terminus after Cysl undergoes its acyl shift. In the presence of thiol reagents, proteins or peptides with carboxy-terminal a-thioesters will spontaneously ligate to polypeptides beginning with cysteine. For synthetic peptides, this process is called native chemical ligation (Dawson et al. 1994); when inteins are involved, the process is called expressed protein ligation (EPL; Muir et al. 1998) or intein-mediated protein ligation (IPL; Evans et al. 1998). Using these techniques, proteins can be stitched together using synthetic or biosynthetic building blocks. Specific regions of proteins can be modified and then ligated to the remaining unmodified portion of the protein. Proteins can be segmentally labeled, phosphorylated, tagged, or synthetic moieties (biosensors, cross-linking reagents, etc.) added, etc. Protein and peptide backbones can be cyclized for increased stability. Cytotoxic proteins can be expressed (Wu et al. 2002). trans-Splicing may reduce the possibility of unwanted gene transfer in transgenic plants (Chin et al. 2003; Yang et al. 2003). Bringing control to a new level, temperature-sensitive inteins were cloned into the transcription regulators Gal4 and Gal80, controlling expression of any gene activated by Gal4 (Zeidler et al. 2004). This system was then used to turn on and off expression of genes regulated by Gal4 in a spatial and temporal fashion in Drosophila embryos by combining temperature-dependent splicing of Gal4 or Gal80 precursors with enhancer-driven tissue-specific expression.
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