Split inteins are protein segments that remove themselves from a larger precursor protein and then rejoin the remaining parts, a process known as protein splicing. They originate from two separate genetic instructions, resulting in two independent fragments: the N-intein and C-intein. These fragments must come together to perform their splicing function, making them valuable tools in biological research.
The Mechanism of Split Intein Splicing
Split intein splicing begins with the independent production of the N-intein and C-intein fragments from separate genes. These fragments are typically fused to the ends of the larger protein segments they will ultimately join, known as exteins. For the splicing reaction to occur, these two intein fragments must physically associate, forming a functional unit. This non-covalent association brings the flanking extein sequences into close proximity.
Once assembled, the split intein initiates an autocatalytic process that does not require external enzymes or energy sources like ATP. The splicing reaction starts with a nucleophilic attack by a specific amino acid, typically a cysteine or serine, at the N-terminus of the intein, on the peptide bond connecting the N-extein and the intein. This attack leads to the formation of a (thio)ester intermediate, linking the N-extein to the intein. A subsequent rearrangement frees the N-terminal end of the intein, forming a branched intermediate where the N-extein and C-extein are temporarily linked, though not by a stable peptide bond.
The final step involves another nucleophilic attack by the free amino group of the C-extein on the (thio)ester bond, which then leads to the excision of the intein fragments. This precise removal of the intein simultaneously results in the formation of a new peptide bond between the N-extein and C-extein, creating a single, mature protein. The excised intein fragments are then released, completing the protein splicing event. The overall process is highly specific, often requiring particular amino acid residues at the splice junctions for efficient activity.
Distinguishing Split Inteins
Split inteins stand apart from single-chain inteins due to their genetic organization. Unlike single-chain inteins, which are encoded by a single continuous gene, split inteins are encoded by two separate genes. This means that the N-intein and C-intein fragments are produced as two distinct polypeptides. These two fragments must then come together in the cell to reconstitute a functional intein capable of catalyzing protein splicing.
The bipartite nature of split inteins offers significant advantages in protein engineering and synthetic biology. Their modularity allows researchers to deliver the N-intein and C-intein fragments independently, potentially from different sources or at different times. This provides flexibility for manipulating protein structure and function, as it enables the joining of two separate proteins or protein fragments that might otherwise be difficult to combine. For instance, one fragment could be produced in a mammalian cell, while the other is synthesized chemically or expressed in bacteria, then joined together by the split intein system.
This “split” design also provides control over the splicing reaction. The interaction and subsequent splicing activity of the split intein fragments can be regulated by various external cues, such as the presence of specific binding partners or environmental conditions. This level of control is harder to achieve with single-chain inteins, where the entire splicing machinery is contained within one polypeptide. Controlling the assembly and activity of split inteins makes them powerful tools for creating complex biological systems and circuits.
Diverse Applications in Biotechnology
Split inteins have become valuable tools in biotechnology, enabling applications in protein engineering and modification. One use is in protein purification, where split inteins can be engineered as self-cleaving tags. A target protein can be fused to an intein segment, and after purification, the intein can be induced to self-cleave, releasing the untagged, pure protein. This method simplifies purification by eliminating the need for proteases or other chemical cleavage agents.
Beyond purification, split inteins are used for site-specific protein labeling and modification. They can facilitate the attachment of molecules, such as fluorescent probes for imaging, or non-natural amino acids, to specific sites within a protein. This is achieved by fusing the intein fragments to the protein of interest and a desired label, then allowing the intein to catalyze the ligation, incorporating the label into the protein. This technique is useful for studying protein dynamics and interactions in live cells.
Split inteins are also employed in protein cyclization, creating stable, circular proteins by joining their N- and C-termini. Circular proteins exhibit increased stability against degradation and thermal resistance compared to their linear counterparts. This is achieved by placing the protein of interest between the two split intein fragments, which then ligate the protein’s ends after excising themselves.
Applications of split inteins extend into biocomputing and living therapeutics. In biocomputing, split inteins can function as molecular “AND” gates, restoring protein function only when both intein fragments are present and can associate to complete splicing. For living therapeutics, split inteins offer a way to overcome limitations in gene delivery, such as the cargo size constraints of adeno-associated viral (AAV) vectors. A large therapeutic protein can be split into two fragments, each delivered by a separate AAV vector, and then reassembled into a functional protein within the target cells by the action of split inteins.