Analysis of Cne Prp8 Intein Substrate Preference Based on mCherry Reporter System

Abstract

Intein-mediated protein splicing provides a valuable tool for protein ligation. The amino acid sequences of the flanking exteins serve as the intein’s natural substrates. Alterations to these extein sequences may cause significant changes in splicing efficiency. To date, how extein sequences at different positions(such as -1 and+2 positions)influence intein splicing activity remains to be systematically investigated. To address this issue, an mCherry-based fluorescence reporter system that links fluorescence intensity to splicing activity was established for high-throughput screening. A saturation mutagenesis library targeting the -1 and+2 positions of the Cne Prp8 intein was constructed via homologous recombination. After induction, 38 high-fluorescence clones were identified from 336 clones. Western blot analysis revealed that three substrate combinations(A/S, S/V, and R/S)achieved splicing efficiencies above 80%, significantly higher than that of the wild-type sequence. These findings not only reveal amino acid preferences at key extein positions but also provide valuable insights for the rational design of efficient splicing systems, thereby advancing the application of inteins in protein engineering.

Keywords

intein / extein / protein splicing / fluorescent protein

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Funding

National Natural Science Foundation of China(31470836)