Protein trans-splicing and its use in structural biology: opportunities and limitations

Obtaining insights into the molecular structure and dynamics of a protein by NMR spectroscopy and other in-solution biophysical methods relies heavily on the incorporation of isotopic labels or other chemical modifications such as fluorescent groups into the protein of interest. These types of modifications can be elegantly achieved with the use of split inteins in a site- and/or region-specific manner. Split inteins are split derivatives of the protein splicing element intein, and catalyze the formation of a peptide bond between two proteins. Recent progress in split intein engineering provided the opportunity to also perform peptide bond formation between a protein and a chemically synthesized peptide. We review the current state-of-the-art in preparing segmental isotope-labeled proteins for NMR spectroscopy, and highlight the importance of split intein orthogonality for the ligation of a protein from multiple fragments. Furthermore, we use split intein-mediated site-specific fluorescent labeling as a framework to illustrate the general usefulness of split inteins for custom protein modifications in the realm of structural biology. We also address some limitations of split intein technology, and offer constructive advice to overcome these shortcomings.