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Updated: Jul 3, 2026
15:06
Synthesis of an Intein-mediated Artificial Protein Hydrogel
Published on: January 27, 2014
Development of a tandem protein trans-splicing system based on native and engineered split inteins
1Laboratory of Synthetic Protein Chemistry, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA.
Journal of the American Chemical Society
|April 28, 2005
Summary
Split inteins enable protein trans-splicing for biotechnology. This study reveals rapid fragment association driven by electrostatics, enabling a novel tandem system for labeling large proteins.
Area of Science:
- Biochemistry
- Molecular Biology
- Biotechnology
Background:
- Protein trans-splicing utilizes split inteins to ligate polypeptides.
- Understanding the molecular recognition of split intein fragment association is limited.
- Split inteins have diverse applications in chemical biology and biotechnology.
Purpose of the Study:
- To investigate the molecular recognition governing split intein fragment association.
- To quantify the binding affinity and kinetics of the Ssp DnaE split intein interaction.
- To develop a novel tandem trans-splicing system for protein assembly and labeling.
Main Methods:
- Fluorescence-based assays were employed to measure binding affinity (dissociation constant).
- On and off rates of fragment association were determined.
- A tandem trans-splicing system was engineered using native and modified split inteins.
Main Results:
- The Ssp DnaE split intein fragments exhibit low nanomolar binding affinity.
- Electrostatic interactions significantly contribute to rapid fragment association at physiological pH.
- A one-pot tandem trans-splicing system was successfully developed for assembling three polypeptides.
Conclusions:
- Split intein fragment association is rapid and electrostatically driven.
- The developed tandem trans-splicing system facilitates efficient protein assembly under native conditions.
- This technology offers a convenient method for segmental labeling of large multidomain proteins.
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