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Related Experiment Video

Updated: Dec 26, 2025

Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

Published on: June 19, 2015

Identification, Characterization, and Optimization of Split Inteins.

Neel H Shah¹, Adam J Stevens²

¹Department of Chemistry, Columbia University, New York, NY, USA. neel.shah@columbia.edu.

Methods in Molecular Biology (Clifton, N.J.)

|March 8, 2020

Summary

This summary is machine-generated.

Split inteins are crucial for protein ligation technologies. New assays using E. coli selection and RP-HPLC offer robust methods to characterize split intein activity for improved protein chemistry.

Keywords:

Bacterial selection Chemical biology Kinetic analysis Protein engineering Protein trans-splicing RP-HPLC Split intein

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Area of Science:

Biochemistry
Molecular Biology
Protein Engineering

Background:

Split inteins are vital tools in peptide and protein chemistry, particularly for protein ligation.
Developing robust split inteins requires methods to assess their activity, stability, and solubility.

Purpose of the Study:

To present two novel assays for characterizing and comparing the activities of split inteins.
To provide tools for identifying and engineering split inteins with enhanced properties.

Main Methods:

An E. coli-based selection system for high-throughput screening of split intein activity.
Reverse-phase high-performance liquid chromatography (RP-HPLC) for detailed analysis of protein splicing kinetics.

Main Results:

The E. coli system enables rapid screening and can be adapted for directed evolution of split inteins.
RP-HPLC provides mechanistic insights into the protein splicing reaction, aiding engineering efforts.
These methods offer alternatives to traditional SDS-PAGE analysis.

Conclusions:

The described assays are valuable for the characterization and engineering of split inteins.
These techniques advance the development of split intein-based protein chemistry technologies.