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An artificially split class 3 intein.

Tia M Ariagno1, John S Smetana1, Christopher W Lennon1

  • 1Department of Biological Sciences, Murray State University, Murray, Kentucky, United States.

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|October 9, 2023
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Summary
This summary is machine-generated.

This study shows protein trans-splicing (PTS) is possible with a split class 3 intein. This offers a more compact catalytic arrangement for splicing compared to split class 1 inteins.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Chemistry

Background:

  • Inteins mediate protein splicing, joining polypeptide fragments (exteins) via a peptide bond.
  • Split inteins are engineered to perform protein trans-splicing (PTS) when expressed as separate fragments.
  • Class 3 inteins possess unique catalytic mechanisms involving an internal nucleophile.

Purpose of the Study:

  • To investigate the feasibility of using an artificially split class 3 intein for protein trans-splicing (PTS).
  • To compare the catalytic arrangement of split class 3 inteins with split class 1 inteins for PTS.

Main Methods:

  • Engineering and expression of an artificially split class 3 intein.
  • Demonstration of protein trans-splicing (PTS) activity in vitro or in vivo.
  • Structural and mechanistic analysis of the split class 3 intein during PTS.

Main Results:

  • Successfully demonstrated PTS using an artificially split class 3 intein.
  • Observed a compact arrangement of catalytic nucleophiles in the split class 3 intein system.
  • This arrangement differs from the standard configuration found in split class 1 inteins.

Conclusions:

  • Artificially split class 3 inteins are effective for protein trans-splicing (PTS).
  • The internal nucleophile of class 3 inteins facilitates a compact catalytic structure for PTS.
  • This finding provides a novel strategy for protein ligation and engineering applications.