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An atypical naturally split intein engineered for highly efficient protein labeling.

Ilka V Thiel1, Gerrit Volkmann, Shmuel Pietrokovski

  • 1Department of Chemistry and Pharmacy, Institute of Biochemistry, University of Münster, Wilhelm-Klemm-Strasse 2, 48149 Münster (Germany).

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PubMed
Summary

A novel, naturally occurring split intein, AceL-TerL, was discovered. Engineered variants offer efficient protein labeling and semi-synthesis, advancing biotechnological applications.

Keywords:
autocatalysischemoenzymatic synthesiscombinatorial mutagenesisprotein engineeringprotein semisynthesis

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Split inteins enable protein trans-splicing for polypeptide backbone assembly.
  • Existing split inteins often lack efficiency or require artificial creation, limiting their use in peptide synthesis.

Purpose of the Study:

  • To identify and characterize novel, naturally occurring split inteins.
  • To develop efficient split inteins for protein labeling and semi-synthesis.

Main Methods:

  • Metagenomic data mining for novel split intein identification.
  • Characterization of the AceL-TerL split intein, including its N-terminal fragment length.
  • Optimization of protein trans-splicing activity through directed evolution and low-temperature conditions.
  • Application in chemical labeling of diverse proteins.

Main Results:

  • Discovery of AceL-TerL, the first naturally occurring, atypically split intein.
  • The N-terminal fragment of AceL-TerL is the shortest natural intein fragment (25 amino acids), suitable for chemical synthesis.
  • Engineered AceL-TerL variants exhibited up to 50-fold increased trans-splicing rates.
  • Demonstrated unprecedented efficiency in chemically labeling various proteins.

Conclusions:

  • AceL-TerL represents a significant advancement in natural split intein discovery.
  • Engineered AceL-TerL variants are highly efficient tools for protein semi-synthesis and chemical labeling.
  • These novel inteins hold great promise for diverse biotechnological applications.