Inteins in Science: Evolution to Application

Abstract

Inteins are mobile genetic elements that apply standard enzymatic strategies to excise themselves post-translationally from the precursor protein via protein splicing. Since their discovery in the 1990s, recent advances in intein technology allow for them to be implemented as a modern biotechnological contrivance. Radical improvement in the structure and catalytic framework of cis- and trans-splicing inteins devised the development of engineered inteins that contribute to various efficient downstream techniques. Previous literature indicates that implementation of intein-mediated splicing has been extended to in vivo systems. Besides, the homing endonuclease domain also acts as a versatile biotechnological tool involving genetic manipulation and control of monogenic diseases. This review orients the understanding of inteins by sequentially studying the distribution and evolution pattern of intein, thereby highlighting a role in genetic mobility. Further, we include an in-depth summary of specific applications branching from protein purification using self-cleaving tags to protein modification, post-translational processing and labelling, followed by the development of intein-based biosensors. These engineered inteins offer a disruptive approach towards research avenues like biomaterial construction, metabolic engineering and synthetic biology. Therefore, this linear perspective allows for a more comprehensive understanding of intein function and its diverse applications.

Production of IgG1-based bispecific antibody without extra cysteine residue via intein-mediated protein trans-splicing

“…Compared to other tags to enable recombination 16 , 19 , 22 , 23 , IMPTS is advantageous in that the third component, an enzyme, is not required, and minimal substrate peptide is left on the product because the Int N -Int C complex is released 21 . Various applications take advantage of these features of IMPTS 20 , 24 – 30 ; BsAbs with the IgG1 structure without the light chain problem have also been developed accordingly (Fig. 1 a) 17 , 18 , 31 .…”

SufB intein splicing inMycobacterium tuberculosisis influenced by two remote conserved N-extein Histidines

“…Splicing and cleavage products (Fig. 1C) are obtained through a series of nucleophilic displacement reactions mediated by the coordinated activity of catalytic residues (5)(6)(7)(8)(9). Therefore, inteins interrupting the functional domain of the host protein, play a regulatory role in protein activation (10,11).…”

“…Especially, the highly conserved Block B His destabilizes the scissile peptide bond either by reducing the energy barrier or by loss of resonance or protonation of the Cys1 amide bond via His imidazole ring to catalyze the N-S acyl shift (14)(15)(16)(17)(18). Block F Asp plays a pivotal role in driving the thioesterification and stabilizing the tetrahedral intermediate by ground-state destabilization (8,16,19). It is also proposed that Block B histidine plays a dual catalytic role;…”

“…The Block A residues Cys, Ser, or Thr participate in the first step of splicing with significant assistance from the Block B His and Thr residues. The highly conserved Block B His destabilizes the scissile peptide bond either by reducing the energy barrier or by loss of resonance or protonation of the Cys1 amide bond via His imidazole ring to catalyze the N-S acyl shift (14)(15)(16)(17)(18).The Block F Asp residue drives the thioesterification through the tetrahedral intermediate by ground-state destabilization (8,16,19). It is also proposed that Block B histidine plays a dual catalytic role; being weakly basic it deprotonates the Cys1 to accelerate the N-S acyl shift and subsequently acts as an acid to stabilize the tetrahedral intermediate (15,17,18).…”

Mini-intein Structures from Extremophiles Suggest a Strategy for Finding Novel Robust Inteins

“…Utilizing the protein splicing activities of inteins bears a repertoire of potential applications in several areas, including in vivo protein engineering, protein purification, and modification. Indeed, intein-mediated chemical reactions have increasingly been incorporated as practical tools in the fields of protein engineering, synthetic biology, and biotechnology [10,11]. For example, inteins from extremely halophilic archaea have been demonstrated to control protein-splicing reaction with salt concentrations, which has enabled the engineering of a salt-inducible self-cleaving tag for protein purification [12].…”