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Salt-sensitive intein for large-scale polypeptide production.

Yi-Zong Lee¹, Shih-Che Sue²

¹Instrument Center, National Tsing Hua University, Hsinchu, Taiwan; Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.

Methods in Enzymology

|May 27, 2019

Summary

This summary is machine-generated.

This study introduces a novel salt-sensitive intein for enzyme-free polypeptide purification. Changing buffer salt concentration efficiently cleaves the fusion protein, simplifying large-scale protein production.

Keywords:

Enzyme-free Fusion protein Intein Isotope-labeling NMR Polypeptide Protein expression Self-cleavage

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Area of Science:

Biochemistry
Molecular Biology
Protein Engineering

Background:

Inteins are self-splicing proteins capable of N- and C-terminal cleavage.
Mutations can disable N-terminal cleavage, preserving C-terminal self-cleavage for fusion protein applications.
Controlling intein activity is crucial for efficient protein purification strategies.

Purpose of the Study:

To develop and characterize a salt-sensitive intein for enzyme-free fusion protein cleavage.
To demonstrate the utility of this system for large-scale polypeptide preparation.
To highlight the advantages of this method over traditional purification techniques.

Main Methods:

Engineering a mutant intein with retained C-terminal self-cleavage activity.
Conjugating the intein to target polypeptide sequences for enhanced expression.
Utilizing changes in ionic strength (buffer conditions) to trigger intein-mediated cleavage.
Characterizing the salt-sensitivity of the intein's cleavage activity.

Main Results:

A salt-sensitive intein was successfully developed, enabling enzyme-free cleavage.
Cleavage activity is regulated by buffer ionic strength, with low salt favoring the reaction.
The method facilitates efficient separation of the intein from the target sequence.
The process is effective for producing proteins from non-Met codons and isotope-labeled peptides.

Conclusions:

Salt-sensitive inteins offer a cost-effective and efficient method for polypeptide purification.
This enzyme-free approach simplifies protein preparation, particularly for large-scale applications.
The ability to control cleavage via buffer change presents a significant advancement in protein engineering.