Characteristics of protein splicing intrans mediated by a semisynthetic split intein

Abstract

Protein splicing in trans results in the ligation of two protein or peptide segments linked to appropriate intein fragments. We have characterized the trans‐splicing reaction mediated by a naturally expressed, approximately 100‐residue N‐terminal fragment of the Mycobacterium tuberculosis intein and a synthetic peptide containing the 38 C‐terminal intein residues, and found that the splicing reaction was very versatile and robust. The efficiency of splicing was nearly independent of temperature between 4 and 37°C and pH between 6.0 and 7.5, with only a slight decline at pH values as high as 8.5. In addition, there was considerable flexibility in the choice of the C‐terminal intein fragment, no significant difference in protein ligation efficiency being observed between reactions utilizing the N‐terminal fragment and either the naturally expressed 107‐residue C‐terminal portion of the intein, much smaller synthetic peptides, or the 107‐residue C‐terminal intein fragment modified by fusion of a maltose binding protein domain to its N‐terminus. The ability to use different types of the C‐terminal intein fragments and a broad range of reaction conditions make protein splicing in trans a versatile tool for protein ligation. © 2000 John Wiley & Sons, Inc. Biopoly 51: 355–362, 1999

Recent progress in intein research: from mechanism to directed evolution and applications

“…Even the immobilization of proteins to a solid support has been achieved by protein trans-splicing [105]. The short C-terminal intein fragment of the naturally split Ssp DnaE (36 aa) and of the artificially split Mtu RecA intein (38 aa) [106,107] were used in the first total synthesis examples of the intein piece by the solid-phase methodology. In recent years, several artificially split intein systems with shorter fragments of 6-15 aa were generated to circumvent the challenging synthesis of peptides C30-40 aa [48,98,108,109].…”

Development of a Chemoenzymatic-like and Photoswitchable Method for the High-Throughput creation of Protein Microarrays. Application to the Analysis of the Protein/Protein Interactions Involved in the YOP Virulon from Yersinia pestis.

“…26 This process is similar to the protein splicing with the only difference that in this case the intein self-processing domain is split in two fragments (called N-intein and Cintein, respectively). 16,[27][28][29][30] These two intein fragments alone are inactive, however, when they are put together under the appropriate conditions they bind specifically each other yielding a totally functional splicing domain, which it splices itself at the same that ligates both extein sequences. In our approach, one of the fragments (C-intein) is covalently attached to the surface through a small peptide-linker meanwhile the other fragment (N-intein) is fused to the C-terminus of the protein to be attached to the surface.…”

Protein trans‐splicing on an M13 bacteriophage: towards directed evolution of a semisynthetic split intein by phage display

“…The ligation can also be performed enzymatically by reverse proteolysis techniques5, 6 or the sortase‐mediated ligation7. Finally, split inteins join two peptide or protein segments (the N ‐ and C ‐terminal exteins) in the autocatalytic protein trans ‐splicing reaction8–10. The synthetic peptides in these approaches can include one or more chemical moieties and structures of virtually any kind, both in the side chains and backbone.…”

“…As the intein fragments are removed in the course of protein splicing, the reaction can be designed in a traceless way. Several split inteins have now been reported of which one of the intein fragments is of only 6 to 36 amino acids in size8, 10, 18, 19, and thus accessible by SPPS, solid phase peptide synthesis. They are spontaneously active under native conditions without the need for a denaturation and a renaturation step.…”