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Site-specific internal protein labeling through trans-splicing.

Xue Li1, Lu Zhang1, Suyang Wang1

  • 1College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, PR China.

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|July 11, 2021
PubMed
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Researchers developed a new method for site-specific internal protein labeling using two atypical split inteins. This technique enables precise modification of proteins with fluorescent probes and other chemical groups.

Keywords:
Atypical split inteinInternal protein labelingTrans-splicing

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Split inteins are utilized for N-terminal or C-terminal protein labeling.
  • Atypical split inteins offer unique properties for protein modification.

Purpose of the Study:

  • To report a novel site-specific internal protein labeling method.
  • To demonstrate the utility of Ter DnaE3 S11 and Rma DnaB S1 split inteins for internal labeling.

Main Methods:

  • In vitro protein-peptide trans-splicing was performed using engineered protein fragments and a peptide tag.
  • The minimal extein requirement for trans-splicing activity was investigated.
  • Fluorescent probes (FITC) were incorporated into recombinant proteins.

Main Results:

  • Successful protein-peptide trans-splicing was demonstrated between recombinant proteins (MBP, Trx) and a peptide (FLAG tag).
  • At least three native extein amino acids (CKG) are required for detectable trans-splicing.
  • Internal labeling of recombinant proteins with FITC was achieved.

Conclusions:

  • The novel method enables site-specific internal protein labeling using atypical split inteins.
  • This technique is effective for incorporating fluorescent probes and other chemical moieties into proteins.
  • The split inteins can facilitate the addition of diverse chemical groups, including biotin, unnatural amino acids, and drug molecules.