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Simultaneous assembly of two target proteins using split inteins for live cell imaging.

Stanley Wong1, Evan Mills, Kevin Truong

  • 1Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.

Protein Engineering, Design & Selection : PEDS
|December 11, 2012
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Summary

This study demonstrates a novel method for simultaneously assembling two target proteins using the split DnaE intein. This protein splicing technique offers equal stoichiometry and fluorescent reporting for versatile applications in live cell imaging.

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Bimolecular Fluorescence Complementation
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Bimolecular Fluorescence Complementation
08:54

Bimolecular Fluorescence Complementation

Published on: April 15, 2011

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Cell Biology

Background:

  • Inteins are protein elements mediating protein splicing, covalently reassembling precursor fragments.
  • Split inteins are utilized for protein ligation, typically for a single target protein.
  • Intein dimerization offers potential for assembling a second target protein.

Purpose of the Study:

  • To demonstrate the simultaneous assembly of two target proteins using the split DnaE intein from Nostoc punctiforme (NpuDnaE).
  • To showcase the versatility of this strategy in live cell imaging applications.
  • To highlight key attributes such as equal stoichiometry and fluorescent reporting of protein splicing.

Main Methods:

  • Utilized the naturally occurring split DnaE intein (NpuDnaE).
  • Applied the intein dimerization strategy for simultaneous protein assembly.
  • Conducted live cell imaging experiments with various protein fusions.

Main Results:

  • Successfully demonstrated simultaneous assembly of two target proteins, including YFP with mRFP, RhoA GTPase mutant with YFP, and GCaMP2 with mRFP.
  • Confirmed equal stoichiometry of the assembled target proteins.
  • Showcased that fluorescent proteins can report the extent of protein splicing.

Conclusions:

  • The split DnaE intein enables a versatile strategy for simultaneous dual protein assembly.
  • The method provides equal stoichiometry and fluorescent readouts, enhancing its utility.
  • The split GCaMP2 with mRFP system shows promise for tissue-specific Ca(2+) imaging in transgenic organisms, with mRFP potentially correcting motion artifacts.