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Synthesis of an Intein-mediated Artificial Protein Hydrogel

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In Cellulo Protein Semi-Synthesis from Endogenous and Exogenous Fragments Using the Ultra-Fast Split Gp41-1 Intein.

Maniraj Bhagawati¹, Simon Hoffmann¹, Katharina S Höffgen¹

¹Department of Chemistry and Pharmacy, Institute of Biochemistry, University of Münster, Corrensstrasse 36, 48149, Münster, Germany.

Angewandte Chemie (International Ed. in English)

|August 11, 2020

Summary

This summary is machine-generated.

Researchers developed a new method for protein semi-synthesis in live cells using the Gp41-1 split intein. This technique enables precise protein labeling for advanced imaging and tracking of proteins within their native cellular environment.

Keywords:

dSTORM protein splicing protein transduction single-molecule studies split intein

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Area of Science:

Biochemistry
Molecular Biology
Cell Biology

Background:

Protein semi-synthesis in live cells allows studying proteins in their native environment.
Split-intein-mediated protein trans-splicing is a powerful tool but requires more established inteins and protocols.
The Gp41-1 split intein's properties needed characterization for broader application.

Purpose of the Study:

To characterize the association and splicing parameters of the Gp41-1 split intein.
To develop and apply a novel split-intein-based protein labeling technique in live mammalian cells.
To enable super-resolution imaging and single-particle tracking of target proteins with minimal perturbation.

Main Methods:

Characterization of Gp41-1 split intein association and splicing kinetics.
Chemical modification and bead-loading of an intein fragment precursor into mammalian cells.
N- and C-terminal fluorescent labeling of target proteins using short peptide tags.
Nuclear-entrapment strategy to reduce cytosolic fluorescence background.
Application of the technique for super-resolution imaging and single-particle tracking of mitochondrial protein Tom20.

Main Results:

The Gp41-1 split intein demonstrated nanomolar affinity and exceptionally fast splicing rates.
The developed technique successfully labeled target proteins with minimal structural or functional perturbation.
Effective reduction of cytosolic fluorescence background was achieved using nuclear entrapment.
Super-resolution imaging and single-particle tracking of Tom20 in HeLa cells were successfully performed.

Conclusions:

The Gp41-1 split intein is a highly efficient tool for protein trans-splicing in live cells.
The novel split-intein-based labeling technique facilitates advanced imaging of proteins in their native cellular context.
This method offers a valuable platform for future studies in cell biology and protein dynamics.