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Fused Split Inteins: Tools for Introducing Multiple Protein Modifications.

Byung Joon Lim1, Raymond F Berkeley1, Galia T Debelouchina2

  • 1Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA.

Methods in Molecular Biology (Clifton, N.J.)
|March 8, 2020
PubMed
Summary
This summary is machine-generated.

Split inteins from the DnaE cyanobacterial family enable efficient protein production and modification. This method uses a fused DnaE intein Npu for traceless purification and C-terminal protein engineering, simplifying complex protein preparation.

Keywords:
Amber suppressionInteinProtein modificationTagless protein purificationUbiquitinUnnatural amino acid incorporation

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Area of Science:

  • Protein Engineering
  • Chemical Biology
  • Biochemistry

Background:

  • Split inteins, particularly from the DnaE cyanobacterial family, are powerful tools for protein engineering.
  • Their rapid splicing kinetics facilitate the in vitro and in vivo production of native proteins from separate polypeptide chains.
  • These inteins can also generate proteins with C-terminal thioesters for subsequent bioconjugation.

Purpose of the Study:

  • To describe a novel method for preparing doubly modified proteins using a genetically fused DnaE intein Npu.
  • To provide protocols for recombinant production of modified ubiquitin, utilizing the fused intein for purification and C-terminal modification.
  • To demonstrate the intein's utility as a traceless purification tag and a tool for introducing C-terminal modifications.

Main Methods:

  • Utilized a genetically fused version of the DnaE intein Npu for recombinant protein expression.
  • Employed amber suppression for the production of modified ubiquitin.
  • Developed a purification protocol that separates truncated products without requiring protease cleavage sites.

Main Results:

  • Successfully produced modified ubiquitin using the fused DnaE intein Npu.
  • Demonstrated the fused intein's effectiveness as a traceless purification tag, simplifying protein separation.
  • Showcased the intein's capability to introduce C-terminal modifications for further protein attachment.

Conclusions:

  • The described method offers a facile and efficient approach for preparing doubly modified proteins.
  • The fused DnaE intein Npu serves as a versatile tool for both protein purification and engineering.
  • This adaptable strategy is suitable for various proteins and applications requiring simultaneous internal and C-terminal modifications.