Substrate specificities of inteins investigated by QuickDrop‐cassette mutagenesis

Abstract

Inteins catalyze self-excision from host precursor proteins while concomitantly ligating the flanking substrates (exteins) with a peptide bond. Noncatalytic extein residues near the splice junctions, such as the residues at the À1 and +2 positions, often strongly influence the protein-splicing efficiency. The substrate specificities of inteins have not been studied for many inteins. We developed a convenient mutagenesis platform termed "QuickDrop"-cassette mutagenesis for investigating the influences of 20 amino acid types at the À1 and +2 positions of different inteins. We elucidated 17 different profiles of the 20 amino acid dependencies across different inteins. The substrate specificities will accelerate our understanding of the structure-function relationship at the splicing junctions for broader applications of inteins in biotechnology and molecular biosciences.

An Atypical Mechanism of Split Intein Molecular Recognition and Folding

“…10 The structure of Cat is similar to that of DnaE inteins, such as Npu (PDB ID: 2KEQ, RMSD 1.45 Å over 92 aligned Cα atoms) and Ssp (PDB ID: 1ZDE, RMSD 1.34 Å over 90 aligned Cα atoms) with the notable exception that Npu and Ssp have an additional helix, which is absent in Cat. 12,25,26 In the Cat active site, a serine residue (Ser 75 ) replaces the threonine located in the canonical TXXH B-block motif (Figure S3c). 27,28 The carbonyl oxygen of C1A is proximal to the amide proton (2.4 Å) and the hydroxyl proton (3.7 Å) of Ser 75 (Figure 4c).…”

“…strain PCC6803 (Ssp) and Nostoc Punctiforme (Npu), fragment assembly was shown to exploit a disorder to order structural transition to produce the topologically intertwined Hedgehog/Intein (HINT) domain. 7,8,12 Alternatively, a split intein from the DNA polymerase of Nanoarchaeum equitans (Neq) employs an extensive structural rearrangement of a pre-folded Neq N-intein upon C-intein interaction. 13 Each of these two mechanisms provides precise control of assembly through coordinated structural rearrangements.…”

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

“…Design of extein in the present study offers a competitive choice under the strategy to maintain the number of Cys in hinge. Local sequences at around junction between intein and extein is the determinant of the reaction efficiency as known for protein splicing 32 – 36 , 43 , but both N- and C-cleaved side products were present at more than negligible level in all the three reactions. It has been reported that large extein protein could promote cleavage side reactions 38 , and the use of large extein (Fab and Fc) in combination with large MBP on the other side of intein might be disadvantageous.…”

The Inducible Intein-Mediated Self-Cleaving Tag (IIST) System: A Novel Purification and Amidation System for Peptides and Proteins

“…Interestingly, the higher concentration of 2 M NaCl showed worse cleavage than the 0.5 M NaCl for exenatide-IIST. Despite the long incubation with the optimized salt condition, the cleavage of the tag was incomplete, suggesting that the junction amino-acid type of “Ser” might influence cleavage efficiency as reported for the IMPACT™ system ( Figure 5 e) [ 5 , 28 , 29 ].…”

“…The Hut MCM2 intein has the wild-type Arg at the so-called -1 position [ 12 , 16 ]. The protein-splicing activity of the Hut MCM2 intein was the best when the -1 position was Arg in the model system [ 29 ]. We estimated the cleavage efficiency of the GLP-1 analog to be >80% after 18 h ( Figure 6 b).…”