A streamlined method for preparing split intein for NMR study

• The method incorporates a circular permutation concept to efficiently prepare native split intein.
• The method avoids the problem of aggregation of intein fragments and is insensitive to the ratio of the two intein fragments.
• The method is applicable to other native and artificial split inteins with various split sites.
• The assignment of NMR spectra of a native split intein is achieved.

A protein ligase, intein, mediates a protein-splicing reaction. It can be split into two complementary fragments and reconstituted as a whole intein scaffold to perform protein trans-splicing. To understand the association of intein fragments and the splicing mechanism, it is necessary to produce a large quantity of split intein for structural study. Conventionally, two fragments are prepared separately and assembled in solution, but severe aggregation of intein fragments occurs, and precise control of the relative concentration of each fragment is difficult. Here, we present a streamlined method to incorporate a circular permutation concept into the production of split intein. By circular permutation of the native split Nostoc punctiforme DnaE intein (NpuInt), a new backbone opening is relocated to the native split site at residue 102. As the protein splicing activity is preserved, the expressed NpuInt can immediately self-cleave into a two-piece split NpuInt. Because of a tight association between the two complementary fragments, split NpuInt can be purified in one step. The idea is simple and applicable to other split inteins. Employing the new preparation, we use NMR spectra to assign the backbone and side chain resonances for the native split NpuInt.