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A redox trap to augment the intein toolbox.

Brian P Callahan¹, Matthew Stanger, Marlene Belfort

¹Department of Biological Sciences, University at Albany, Life Sciences Building 2061, 1400 Washington Avenue, Albany, New York 12222, USA. callahan@binghamton.edu

Biotechnology and Bioengineering

|January 3, 2013

Summary

This summary is machine-generated.

Researchers developed a redox-controlled intein system for biotechnology. This disulfide trap regulates intein activity, enhancing protein ligation, bioseparation, and enabling a novel bacterial redox sensor.

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Area of Science:

Biochemistry
Molecular Biology
Biotechnology

Background:

Intein activity is often unregulated during expression and work-up, limiting their biotechnological applications.
Intein-mediated protein ligation (IMPL) and bioseparation methods can be inefficient due to side reactions.
Controlling intein catalysis is crucial for expanding their use in chemical biology.

Purpose of the Study:

To develop a mechanism-based approach to regulate intein autocatalysis for biotechnological applications.
To engineer a redox-controllable intein system using a disulfide bond to trap the catalytic cysteine residue.
To demonstrate the utility of this redox-trap intein technology in protein ligation, bioseparation, and bacterial sensing.

Main Methods:

Utilized standard mutagenesis to introduce a disulfide bond for reversible trapping of the intein's catalytic cysteine.
Applied the disulfide trap to various inteins for redox control in vitro and in Escherichia coli.
Developed a novel bacterial redox sensor based on the disulfide-trap intein technology.

Main Results:

Enhanced precursor recovery and product yield by fourfold to sixfold in intein-mediated protein ligation (expressed protein ligation).
Achieved twofold improvement in precursor recovery and product yield in bioseparation experiments.
Successfully developed a bacterial redox sensor capable of distinguishing hyperoxic E. coli with disrupted reductive pathways from wild-type cells.

Conclusions:

The disulfide-trap intein system provides effective redox control over intein activity, overcoming limitations of unregulated inteins.
This technology significantly improves yields and efficiency in protein ligation and bioseparation applications.
The developed bacterial redox sensor offers a novel tool for monitoring cellular redox states and identifying specific genetic mutations.