Intein applications: from protein purification and labeling to metabolic control methods

Abstract

The discovery of inteins in the early 1990s opened the door to a wide variety of new technologies. Early engineered inteins from various sources allowed the development of self-cleaving affinity tags and new methods for joining protein segments through expressed protein ligation. Some applications were developed around native and engineered split inteins, which allow protein segments expressed separately to be spliced together in vitro. More recently, these early applications have been expanded and optimized through the discovery of highly efficient trans-splicing and trans-cleaving inteins. These new inteins have enabled a wide variety of applications in metabolic engineering, protein labeling, biomaterials construction, protein cyclization, and protein purification.

Keywords: Intein; Protein Chemical Modification; Protein Cyclization; Protein Engineering; Protein Labeling; Protein Processing; Protein Purification; Protein Self-assembly; Protein Splicing; Self-cleaving Tag.

FIGURE 1.

Self-cleaving affinity tags based on inteins. A, initial tags used thiol addition or temperature (Temp) and/or pH changes to induce cleaving in cis-cleaving inteins. The IMPACT-CN system (left) includes an affinity tag within the intein, and C-terminal cleaving is induced via thiol-induced cleavage of a short N-terminal extein peptide (step 1 at bottom). Cleavage of N-extein peptide then leads to rapid C-terminal cleavage (step 2 at bottom), and the N-terminal peptide is subsequently separated from the cleaved target protein by dialysis. The ΔI-CM intein (right) provides C-terminal cleaving in the absence of N-terminal cleavage, where the cleavage reaction is controlled by shifts in pH and/or temperature. In this intein, the cleavage reaction is additionally accelerated through mutation of a conserved aspartic acid to glycine, close to the C terminus of the intein. B, the Ssp DnaB intein has been engineered with an 11-residue deletion at its N terminus to eliminate premature cleaving during expression. C-terminal cleavage of the intein can be induced by the addition of the 11-residue intein segment (left panel), or conversely, N-terminal cleaving from the 11-residue intein segment can be induced by the addition of the remaining intein in the presence of thiol (right panel). C, the Npu DnaE naturally split intein has been engineered with an internal affinity tag to provide extraordinarily rapid cleaving upon reassembly. This intein effectively combines the N-extein removal of the IMPACT-CN system with the aspartic acid to glycine mutation of the ΔI-CM intein, leading to very rapid cleaving that can be controlled by intein reassembly in the presence of zinc.

FIGURE 2.

Intein applications involving post-translational modifications of target proteins. A, left panel, EPL methods involve a nucleophilic attack of an N-terminal Cys residue on a thioester formed by a downstream intein. The N-terminal Cys can be generated by a second, upstream intein or by conventional proteolytic cleavage. Right panel, PTS methods produce cyclized proteins through the assembly and splicing of an inverted split intein fused to the N and C terminus of the target protein. B, fluorescent labeling of proteins using a self-quenched intein-based PTS reagent. In this case, PTS simultaneously labels the target protein while releasing the quencher from the dye, thus providing a strong label signal with minimal background from unreacted label. C, protein-protein interactions can be detected by fusing “bait” and “prey” proteins to each half of a weakly interacting trans-splicing intein. Interactions between bait and prey drive assembly and splicing of the intein, resulting in activation of a reporter enzyme.